Compounds and pharmaceutical compositions for the treatment of viral infections

ABSTRACT

Provided herein are compounds, compositions and methods for the treatment of liver disorder, including HCV and/or HBV infections. Specifically, compound and compositions of nucleoside derivatives are disclosed, which can be administered either alone or in combination with other anti-viral agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/005,937, filed on Dec. 27, 2007, now U.S. Pat. No. 7,951,789 whichclaims of priority to 1) U.S. Provisional Appl. No. 60/877,944, filedDec. 28, 2006; 2) U.S. Provisional Appl. No. 60/936,290, filed Jun. 18,2007; and 3) U.S. Provisional Application No. 60/985,891, filed Nov. 6,2007. The disclosures of the above referenced applications areincorporated by reference in their entireties herein.

FIELD

Provided herein are compounds, methods and pharmaceutical compositions,for use in treatment of viral infections, including hepatitis C virusinfection, and hepatitis B virus infection in a host in need thereof. Ina particular embodiment, phosphoroamidate or phosphonoamidate nucleosidecompounds are provided which allow concentration of the drug in theliver.

BACKGROUND

Flaviviridae Viruses

The Flaviviridae family of viruses comprises at least three distinctgenera: pestiviruses, which cause disease in cattle and pigs;flaviviruses, which are the primary cause of diseases such as denguefever and yellow fever; and hepaciviruses, whose sole member is HCV. Theflavivirus genus includes more than 68 members separated into groups onthe basis of serological relatedness (Calisher et al., J. Gen. Virol,1993, 70, 37-43). Clinical symptoms vary and include fever, encephalitisand hemorrhagic fever (Fields Virology, Editors: Fields, B. N., Knipe,D. M., and Howley, P. M., Lippincott-Raven Publishers, Philadelphia,Pa., 1996, Chapter 31, 931-959). Flaviviruses of global concern that areassociated with human disease include the dengue hemorrhagic feverviruses (DHF), yellow fever virus, shock syndrome and Japaneseencephalitis virus (Halstead, S. B., Rev. Infect. Dis., 1984, 6,251-264; Halstead, S. B., Science, 239:476-481, 1988; Monath, T. P., NewEng. J. Med., 1988, 319, 641-643).

The pestivirus genus includes bovine viral diarrhea virus (BVDV),classical swine fever virus (CSFV, also called hog cholera virus) andborder disease virus (BDV) of sheep (Moennig, V. et al. Adv. Vir. Res.1992, 41, 53-98). Pestivirus infections of domesticated livestock(cattle, pigs and sheep) cause significant economic losses worldwide.BVDV causes mucosal disease in cattle and is of significant economicimportance to the livestock industry (Meyers, G. and Thiel, H.-J.,Advances in Virus Research, 1996, 47, 53-118; Moennig V., et al, Adv.Vir. Res. 1992, 41, 53-98). Human pestiviruses have not been asextensively characterized as the animal pestiviruses. However,serological surveys indicate considerable pestivirus exposure in humans.

Pestiviruses and hepaciviruses are closely related virus groups withinthe Flaviviridae family. Other closely related viruses in this familyinclude the GB virus A, GB virus A-like agents, GB virus-B and GBvirus-C (also called hepatitis G virus, HGV). The hepacivirus group(hepatitis C virus; HCV) consists of a number of closely related butgenotypically distinguishable viruses that infect humans. There areapproximately 6 HCV genotypes and more than 50 subtypes. Due to thesimilarities between pestiviruses and hepaciviruses, combined with thepoor ability of hepaciviruses to grow efficiently in cell culture,bovine viral diarrhea virus (BVDV) is often used as a surrogate to studythe HCV virus.

The genetic organization of pestiviruses and hepaciviruses is verysimilar. These positive stranded RNA viruses possess a single large openreading frame (ORF) encoding all the viral proteins necessary for virusreplication. These proteins are expressed as a polyprotein that is co-and post-translationally processed by both cellular and virus-encodedproteinases to yield the mature viral proteins. The viral proteinsresponsible for the replication of the viral genome RNA are locatedwithin approximately the carboxy-terminal. Two-thirds of the ORF aretermed nonstructural (NS) proteins. The genetic organization andpolyprotein processing of the nonstructural protein portion of the ORFfor pestiviruses and hepaciviruses is very similar. For both thepestiviruses and hepaciviruses, the mature nonstructural (NS) proteins,in sequential order from the amino-terminus of the nonstructural proteincoding region to the carboxy-terminus of the ORF, consist of p7, NS2,NS3, NS4A, NS4B, NS5A, and NS5B.

The NS proteins of pestiviruses and hepaciviruses share sequence domainsthat are characteristic of specific protein functions. For example, theNS3 proteins of viruses in both groups possess amino acid sequencemotifs characteristic of serine proteinases and of helicases (Gorbalenyaet al. (1988) Nature 333:22; Bazan and Fletterick (1989) Virology171:637-639; Gorbalenya et al. (1989) Nucleic Acid Res. 17.3889-3897).Similarly, the NS5B proteins of pestiviruses and hepaciviruses have themotifs characteristic of RNA-directed RNA polymerases (Koonin, E. V. andDolja, V. V. (1993) Crit. Rev. Biochem. Molec. Biol. 28:375-430).

The actual roles and functions of the NS proteins of pestiviruses andhepaciviruses in the lifecycle of the viruses are directly analogous. Inboth cases, the NS3 serine proteinase is responsible for all proteolyticprocessing of polyprotein precursors downstream of its position in theORF (Wiskerchen and Collett (1991) Virology 184:341-350; Bartenschlageret al. (1993) J. Virol. 67:3835-3844; Eckart et al. (1993) Biochem.Biophys. Res. Comm. 192:399-406; Grakoui et al. (1993) J. Virol.67:2832-2843; Grakoui et al. (1993) Proc. Natl. Acad. Sci. USA90:10583-10587; Hijikata et al. (1993) J. Virol. 67:4665-4675; Tome etal. (1993) J. Virol. 67:4017-4026). The NS4A protein, in both cases,acts as a cofactor with the NS3 serine protease (Bartenschlager et al.(1994) J. Virol. 68:5045-5055; Failla et al. (1994) J. Virol. 68:3753-3760; Lin et al. (1994) 68:8147-8157; Xu et al. (1997) J. Virol.71:5312-5322). The NS3 protein of both viruses also functions as ahelicase (Kim et al. (1995) Biochem. Biophys. Res. Comm. 215: 160-166;Jin and Peterson (1995) Arch. Biochem. Biophys., 323:47-53; Warrener andCollett (1995) J. Virol. 69:1720-1726). Finally, the NS5B proteins ofpestiviruses and hepaciviruses have the predicted RNA-directed RNApolymerases activity (Behrens et al. (1996) EMBO J. 15:12-22; Lehmann etal. (1997) J. Virol. 71:8416-8428; Yuan et al. (1997) Biochem. Biophys.Res. Comm. 232:231-235; Hagedorn, PCT WO 97/12033; U.S. Pat. Nos.5,981,247; 6,248,589 and 6,461,845 Zhong et al. (1998) J. Virol.72.9365-9369).

Hepatitis C Virus

The hepatitis C virus (HCV) is the leading cause of chronic liverdisease worldwide. (Boyer, N. et al. J. Hepatol. 32:98-112, 2000). HCVcauses a slow growing viral infection and is the major cause ofcirrhosis and hepatocellular carcinoma (Di Besceglie, A. M. and Bacon,B. R., Scientific American, October: 80-85, (1999); Boyer, N. et al. J.Hepatol. 32:98-112, 2000). An estimated 170 million persons are infectedwith HCV worldwide. (Boyer, N. et al. J. Hepatol. 32:98-112, 2000).Cirrhosis caused by chronic hepatitis C infection accounts for8,000-12,000 deaths per year in the United States, and HCV infection isthe leading indication for liver transplantation.

HCV is known to cause at least 80% of posttransfusion hepatitis and asubstantial proportion of sporadic acute hepatitis. Preliminary evidencealso implicates HCV in many cases of “idiopathic” chronic hepatitis,“cryptogenic” cirrhosis, and probably hepatocellular carcinoma unrelatedto other hepatitis viruses, such as Hepatitis B Virus (HBV). A smallproportion of healthy persons appear to be chronic HCV carriers, varyingwith geography and other epidemiological factors. The numbers maysubstantially exceed those for HBV, though information is stillpreliminary; how many of these persons have subclinical chronic liverdisease is unclear. (The Merck Manual, ch. 69, p. 901, 16th ed.,(1992)).

HCV is an enveloped virus containing a positive-sense single-strandedRNA genome of approximately 9.4 kb. The viral genome consists of a 5′untranslated region (UTR), a long open reading frame encoding apolyprotein precursor of approximately 3011 amino acids, and a short 3′UTR. The 5′ UTR is the most highly conserved part of the HCV genome andis important for the initiation and control of polyprotein translation.Translation of the HCV genome is initiated by a cap-independentmechanism known as internal ribosome entry. This mechanism involves thebinding of ribosomes to an RNA sequence known as the internal ribosomeentry site (IRES). An RNA pseudoknot structure has recently beendetermined to be an essential structural element of the HCV IRES. Viralstructural proteins include a nucleocapsid core protein (C) and twoenvelope glycoproteins, E1 and E2. HCV also encodes two proteinases, azinc-dependent metalloproteinase encoded by the NS2-NS3 region and aserine proteinase encoded in the NS3 region. These proteinases arerequired for cleavage of specific regions of the precursor polyproteininto mature peptides. The carboxyl half of nonstructural protein 5,NS5B, contains the RNA-dependent RNA polymerase. The function of theremaining nonstructural proteins, NS4A and NS4B, and that of NS5A (theamino-terminal half of nonstructural protein 5) remain unknown.

A significant focus of current antiviral research is directed to thedevelopment of improved methods of treatment of chronic HCV infectionsin humans (Di Besceglie, A. M. and Bacon, B. R., Scientific American,October: 80-85, (1999)).

In light of the fact that HCV infection has reached epidemic levelsworldwide, and has tragic effects on the infected patient, there remainsa strong need to provide new effective pharmaceutical agents to treathepatitis C that have low toxicity to the host.

Further, given the rising threat of other flaviviridae infections, thereremains a strong need to provide new effective pharmaceutical agentsthat have low toxicity to the host.

Hepatitis B

Hepatitis B virus has reached epidemic levels worldwide. After a two tosix month incubation period in which the host is unaware of theinfection, HBV infection can lead to acute hepatitis and liver damage,that causes abdominal pain, jaundice, and elevated blood levels ofcertain enzymes. HBV can cause fulminant hepatitis, a rapidlyprogressive, often fatal form of the disease in which massive sectionsof the liver are destroyed. Patients typically recover from acute viralhepatitis. In some patients, however, high levels of viral antigenpersist in the blood for an extended, or indefinite, period, causing achronic infection. Chronic infections can lead to chronic persistenthepatitis. Patients infected with chronic persistent HBV are most commonin developing countries. Chronic persistent hepatitis can cause fatigue,cirrhosis of the liver and hepatocellular carcinoma, a primary livercancer. In western industrialized countries, high risk groups for HBVinfection include those in contact with HBV carriers or their bloodsamples. The epidemiology of HBV is in fact very similar to that ofacquired immunodeficiency syndrome, which accounts for why HBV infectionis common among patients with AIDS or HIV-associated infections.However, HBV is more contagious than HIV.

Daily treatments with α-interferon, a genetically engineered protein,have shown promise. A human serum-derived vaccine has also beendeveloped to immunize patients against HBV. Vaccines have been producedthrough genetic engineering. While the vaccine has been found effective,production of the vaccine is troublesome because the supply of humanserum from chronic carriers is limited, and the purification procedureis long and expensive. Further, each batch of vaccine prepared fromdifferent serum must be tested in chimpanzees to ensure safety. Inaddition, the vaccine does not help the patients already infected withthe virus.

An essential step in the mode of action of purine and pyrimidinenucleosides against viral diseases, and in particular, HBV and HCV istheir metabolic activation by cellular kinases, to yield the mono-, di-and triphosphate derivatives. The biologically active species of manynucleosides is the triphosphate form, which inhibits viral DNApolymerase, RNA polymerase, or reverse transcriptase, or causes chaintermination.

In light of the fact that hepatitis B and C viruses have reachedepidemic levels worldwide, and has severe and often tragic effects onthe infected patient, there remains a strong need to provide neweffective pharmaceutical agents to treat humans infected with the virusthat have low toxicity to the host.

Therefore, there is a continuing need for effective treatments of HCVand HBV infections.

SUMMARY

Phosphoramidate and phosphonoamidate compounds of a variety oftherapeutic agents are provided, as well as methods for theirmanufacture and use in the treatment of a variety of disorders includingliver disorders. Such compounds can be used in some embodiments topermit concentration of the therapeutic agent in the liver. In oneembodiment, the compound is a S-pivaloyl-2-thioethyl phosphoramidate,S-pivaloyl-2-thioethyl phosphonoamidate, S-hydroxypivaloyl-2-thioethylphosphoramidate or S-hydroxypivaloyl-2-thioethyl phosphonoamidate.

Phosphoramidate or phosphonoamidate compounds of a variety oftherapeutic agents are provided. As used herein, a “phosphoramidate orphosphonoamidate compound of a therapeutic agent” includes a therapeuticagent derivatized to include a phosphoramidate or phosphonoamidategroup. The therapeutic agent is, for example, an anti-viral agent thatincludes, or has been derivatized to include, a reactive group, such asa hydroxyl, for attachment of the phosphoramidate or phosphonoamidatemoiety. Such therapeutic agents include, but are not limited tonucleosides and nucleoside analogs including acyclic nucleosides. Insome embodiments, phosphoramidates of nucleotides and nucleotide analogsare also provided, such as phosphoramidates of 1′, 2′, 3′-branched and4′-branched nucleosides. Such compounds can be administered in aneffective amount for the treatment of liver disorders, includinginfectious diseases, such as hepatitis B and hepatitis C infection,including resistant strains thereof.

In certain embodiments, while not being limited to any theory, it ispossible that the parent drug is obtained from selective metabolism ofthe phosphoramidate or phosphonoamidate compound in the liver, and thusthe parent drug is capable of accumulating in the liver of a host. Byselectively targeting and activating compounds in the liver, potentiallyundesired distribution of active compound in the gastrointestinal tractcan be reduced. Moreover, therapeutic amounts of active compound at thesite of infection in the liver can be increased.

In certain embodiments, a 5′-monophosphate or phosphonate of a parentnucleoside (or nucleoside derivative) drug is formed from metabolism ofthe phosphoramidate or phosphonoamidate compound in the liver, allowingthe monophosphate or phosphonate to form and accumulate in the liver ofa host. Thus, in certain embodiments, the phosphoramidate in effectprovides a stabilized phosphate on the nucleoside or nucleosideanalogue. In certain embodiments, where the compound needs to betriphosphorylated to be active, this advantageously can eliminate therequirement for the initial phosphorylation step, and promote more readyformation of the active triphosphate, which inhibits the target enzyme,and can enhance the overall activity of the nucleoside or nucleosideanalog.

Without being limited to any theory, in one embodiment, aphosphoramidate of a nucleoside, such as a 2′-C-methyl-ribonucleoside,is provided, that is selectively concentrated in the liver after oraladministration, and metabolized in the liver cell to yield a5′-monophosphate that can be enzymatically converted to the active formof the 5′-triphosphate, which inhibits the HCV polymerase. Thuspotentially therapeutic doses can be reduced in comparison toadministering the nucleoside parent molecule.

Thus, in some embodiments, after oral administration of thephosphoramidate and phosphonamidate compounds described herein, thecompounds can advantageously concentrate in the liver cells at the siteof infection and convert to the phosphate or phosphonate in the livercell, which then is optionally further phosphorylated to implement itstherapeutic effect.

Since these methods allow accumulation of the phosphoramidate orphosphonoamidate compounds disclosed herein in the liver of a host, themethods described herein can be useful, for example, for the treatmentand/or prophylaxis of diseases or disorders of the liver, such ashepatitis B or C.

In certain embodiments, the compounds provided herein are useful in theprevention and treatment of Flaviviridae infections and other relatedconditions such as anti-Flaviviridae antibody positive andFlaviviridae-positive conditions, chronic liver inflammation caused byHCV, cirrhosis, fibrosis, acute hepatitis, fulminant hepatitis, chronicpersistent hepatitis, and fatigue. These compounds or formulations canalso be used prophylactically to prevent or retard the progression ofclinical illness in individuals who are anti-Flaviviridae antibody orFlaviviridae-antigen positive or who have been exposed to aFlaviviridae. In one specific embodiment, the Flaviviridae is hepatitisC. In certain embodiments, the compound is used to treat any virus thatreplicates through an RNA-dependent RNA polymerase.

A method for the treatment of a Flaviviridae infection in a host,including a human, is also provided that includes administering aneffective amount of a compound provided herein, administered eitheralone or in combination or alternation with another anti-Flaviviridaeagent, optionally in a pharmaceutically acceptable carrier.

In certain embodiments, a method for treatment and/or prophylaxis ofhepatitis B infections and other related conditions such as anti-HBVantibody positive and HBV-positive conditions, chronic liverinflammation caused by HBV, fibrosis, cirrhosis, acute hepatitis,fulminant hepatitis, chronic persistent hepatitis, and fatigue areprovided herein.

In certain embodiments, phosphoramidate or phosphonoamidate compounds ofa variety of pharmaceutical agents can be made and used therapeuticallyas described herein, to enhance delivery of the drug to the liver. Inone embodiment, the compound is an S-acyl-2-thioethyl phosphoramidate oran S-acyl-2-thioethyl phosphonoamidate derivative, e.g., aS-pivaloyl-2-thioethyl phosphoramidate or aS-hydroxypivaloyl-2-thioethyl phosphonoamidate derivative.

The phosphoramidate or phosphonoamidate compounds, as well as saltsthereof, and compositions comprising the compounds, provided herein areuseful for treatment of disorders of the liver, such as HBV and/or HCVinfections. In one embodiment, the compound provided herein is acompound of Formula I:

or a pharmaceutically acceptable salt, solvate, a stereoisomeric,tautomeric or polymorphic form thereof, wherein

X^(a) is

Z is O or S;

each W is independently O or S;

R^(y) and R^(u) each independently represent alkyl, alkenyl, alkynyl,aryl, aryl alkyl, cycloalkyl, cycloalkenyl, amino, aminoalkyl,hydroxyalkyl, alkoxy, heterocyclyl, or heteroaryl, all optionallysubstituted;

R^(a) and R^(b) are selected as follows:

i) R^(a) and R^(b) are each independently hydrogen, alkyl, carboxyalkyl,hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl,alkoxycarbonylalkyl, aryl, aryl alkyl, cycloalkyl, aryl, heteroaryl orheterocyclyl, all optionally substituted; or

ii) R^(a) and R^(b) together with the nitrogen atom on which they aresubstituted form a 3-7 membered heterocyclic or heteroaryl ring;

n is 0-3; n2 is 1-4; and

R¹ is a moiety derivable by removal of a hydrogen from a hydroxy groupof an anti-viral drug.

In another embodiment,

X^(a) is

Z is O, S, NH or NR^(w), where R^(w) is, e.g., alkyl, alkyl, alkenyl,alkynyl, aryl, aryl alkyl, cycloalkyl, cycloalkenyl, amino, aminoalkyl,alkoxy, heterocyclyl, or heteroaryl, all optionally substituted;

each W is O, S, NH or NR^(w), where R^(w) is, e.g., alkyl, alkyl,alkenyl, alkynyl, aryl, aryl alkyl, cycloalkyl, cycloalkenyl, amino,aminoalkyl, alkoxy, heterocyclyl, or heteroaryl, all optionallysubstituted;

R^(y) and R^(u) each independently represent alkyl, alkenyl, alkynyl,aryl, aryl alkyl, cycloalkyl, cycloalkenyl, amino, aminoalkyl, alkoxy,heterocyclyl, or heteroaryl, all optionally substituted;

R^(a) and R^(b) are selected as follows:

i) R^(a) and R^(b) are each independently hydrogen, alkyl, carboxyalkyl,hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl,alkoxycarbonylalkyl, aryl, aryl alkyl, cycloalkyl, heteroaryl orheterocyclyl, all optionally substituted; or

ii) R^(a) and R^(b) together with the nitrogen atom on which they aresubstituted form a 3-7 membered heterocyclic or heteroaryl ring;

n is 0-3; n₂ is 1-4; and

R¹ is as described herein.

Those of skill in the art will recognize that compounds of Formula I canbe designed or prepared by reaction, e.g., at a hydroxy group of saidanti-viral drug, for example, via condensation or dehydration. Forconvenience, in the description herein when substituents, such asexemplary R¹ groups are identified as a drug, those of skill in the artwill recognize that the compound e.g. of Formula I comprises aderivative, e.g. a radical of the anti-viral drug. Those derivatives canfor example be prepared by elimination of a hydrogen radical from ahydroxy group of the drug, for instance in a dehydration reaction. Whereappropriate, certain derivatives can be prepared by modification of aphosphate or phosphonate of an anti-viral drug to yield a compound offormula I.

In certain embodiments of Formula I, R¹ is a nucleoside comprising acyclic or acyclic sugar or an analog thereof.

In certain embodiments, R¹ is an anti-viral nucleoside analog useful fortreatment of HCV virus infection selected from ribavirin, viramidine,2′-C-methylcytidine, 2′-C-methylguanosine, valopicitabine (NM 283),MK-0608 and PSI-6130.

In certain embodiments, R¹ is an anti-viral nucleoside analog useful fortreatment of HBV virus infection selected from lamivudine (Epivir-HBV,Zeffix, or Heptodin), adefovir, entecavir (Baraclude), telbivudine(Tyzeka, Sebivo), emtricitabine (FTC), clevudine (L-FMAU), viread(Tenofovir), torcitabine, valtorcitabine (monoval LdC), amdoxovir (DAPD)and RCV (Racivir).

In certain embodiments, R¹ is a non-nucleoside anti-viral useful fortreatment of HBV virus infection selected from resiquimod or celgosivir.

In certain embodiments according to Formula I, R^(y) is substitutedalkyl, e.g. hydroxyalkyl or aminoalkyl; and R^(a) and R^(b) are eachindependently hydrogen, alkyl, substituted alkyl, benzyl or substitutedbenzyl, for instance hydroxy- or amino-substituted alkyl or benzyl. Inanother embodiment, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c) where eachR^(c) is independently alkyl, substituted alkyl, aryl or substitutedaryl, for instance hydroxy- or amino-substituted alkyl or aryl; andR^(a) and R^(b) are independently hydrogen, alkyl, substituted alkyl,benzyl or substituted benzyl, for instance hydroxy- or amino-substitutedalkyl or benzyl. In a further embodiment, R^(a) and R^(b) areindependently benzyl or substituted alkyl. In a further embodiment,R^(y) is selected from the group consisting of alkyl and hydroxyalkyl.In certain embodiments, R^(y) is —C(CH₃)₂CH₂OH.

In certain embodiments, the compounds provided herein are selected suchthat R¹ is not 3′-azido-2′,3′-dideoxythymidine.

In another embodiment, the compound provided herein is a compound ofFormula IIa or IIb:

or a pharmaceutically acceptable salt, solvate, a stereoisomeric,tautomeric or polymorphic form thereof, wherein

R^(y) is alkyl, alkenyl, alkynyl, aryl, aryl alkyl, cycloalkyl,cycloalkenyl, amino, aminoalkyl, hydroxyalkyl, heterocyclyl orheteroaryl, all optionally substituted;

R^(a) and R^(b) are selected as follows:

i) R^(a) and R^(b) are each independently hydrogen, alkyl, carboxyalkyl,hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl,alkoxycarbonylalkyl, aryl, aryl alkyl, cycloalkyl, aryl, heteroaryl orheterocyclyl, all optionally substituted; or

ii) R^(a) and R^(b) together with the nitrogen atom on which they aresubstituted form a 3-7 membered heterocyclic or heteroaryl ring; and

R¹ is an antiviral drug (as used herein where R¹ is an antiviral drug,that embodiment includes a moiety derivable by removal of a hydrogenfrom a hydroxy group of an anti-viral drug), such as a nucleoside ornucleoside analog.

In certain embodiments according to Formula IIa or IIb, R^(y) issubstituted alkyl, e.g. hydroxyalkyl or aminoalkyl; and R^(a) and R^(b)are independently hydrogen, alkyl, substituted alkyl, benzyl orsubstituted benzyl, for instance hydroxy- or amino-substituted alkyl orbenzyl. In another embodiment, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c)where each R^(c) is independently alkyl, substituted alkyl, aryl orsubstituted aryl, for instance hydroxy- or amino-substituted alkyl oraryl; and R^(a) and R^(b) are independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl, for instance hydroxy- oramino-substituted alkyl or benzyl. In a further embodiment, R^(a) andR^(b) are independently benzyl or substituted alkyl. In a furtherembodiment, R^(y) is selected from the group consisting of alkyl andhydroxyalkyl. In certain embodiments, R^(y) is —C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound ofFormula:

wherein R^(a), R^(b) and R^(y) are as described in Formula I and

wherein R² and R³ are each independently H, straight chained, branchedor cyclic alkyl; acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate estersuch as alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted;alkylsulfonyl, arylsulfonyl, aryl alkylsulfonyl, a lipid, such as aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich is capable of providing a compound wherein R² and/or R³ isindependently H, for example when administered in vivo; or R² and R³ arelinked to form a cyclic group by an alkyl, ester or carbamate linkage;and wherein each R^(L) is independently H, carbamyl, straight chained,branched or cyclic alkyl; acyl (including lower acyl); CO-alkyl,CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonateester such as alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted;alkylsulfonyl, arylsulfonyl, aryl alkylsulfonyl, a lipid, such as aphospholipid; an amino acid; an amino acid residue; or a carbohydrate.In certain embodiments according to this paragraph, R² and R³ are eachH; R^(y) is substituted alkyl, e.g. hydroxyalkyl or aminoalkyl; andR^(a) and R^(b) are independently hydrogen, alkyl, substituted alkyl,benzyl or substituted benzyl, for instance hydroxy- or amino-substitutedalkyl or benzyl. In another embodiment, R² and R³ are each H; R^(y) is—OR^(c), —C(R^(c))₃ or —NHR^(c) where each R^(c) is independently alkyl,substituted alkyl, aryl or substituted aryl, for instance hydroxy- oramino-substituted alkyl or aryl; and R^(a) and R^(b) are independentlyhydrogen, alkyl, substituted alkyl, benzyl or substituted benzyl, forinstance hydroxy- or amino-substituted alkyl or benzyl. In a furtherembodiment, R² and R³ are each H; R^(a) and R^(b) are independentlybenzyl or substituted alkyl. In a further embodiment, R² and R³ are eachH; R^(y) is selected from the group consisting of alkyl andhydroxyalkyl. In certain embodiments, R² and R³ are each H; R^(y) is—C(CH₃)₂CH₂OH. In certain embodiments according to this paragraph, R²and R³ are each hydrogen, R^(a) is hydrogen, R^(b) is —CH₂—C₆H₅ andR^(y) is —C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

wherein R^(a), R^(b) and R^(y) are as described in Formula I. R^(d) isselected from the group consisting of hydrogen, alkyl and alkoxy. Incertain embodiments, R^(d) is hydrogen, methyl or methoxy. In certainembodiments according to this paragraph, R^(y) is substituted alkyl,e.g. hydroxyalkyl or aminoalkyl; and R^(a) and R^(b) are independentlyhydrogen, alkyl, substituted alkyl, benzyl or substituted benzyl, forinstance hydroxy- or amino-substituted alkyl or benzyl. In anotherembodiment, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c) where each R^(c) isindependently alkyl, substituted alkyl, aryl or substituted aryl, forinstance hydroxy- or amino-substituted alkyl or aryl; and R^(a) andR^(b) are independently hydrogen, alkyl, substituted alkyl, benzyl orsubstituted benzyl, for instance hydroxy- or amino-substituted alkyl orbenzyl. In a further embodiment, R^(a) and R^(b) are independentlybenzyl or substituted alkyl. In a further embodiment, R^(y) is selectedfrom the group consisting of alkyl and hydroxyalkyl. In certainembodiments, R^(y) is —C(CH₃)₂CH₂OH. In certain embodiments according tothis paragraph, R² and R³ are each hydrogen, R^(a) is hydrogen, R^(b) is—CH₂—C₆H₅ and R^(y) is —C(CH₃)₂CH₂OH In certain embodiments according tothis paragraph, R^(a) is hydrogen, R^(b) is —CH₂—C₆H₅ and R^(y) is—C(CH₃)₂CH₂OH.

In one embodiment, the nucleosides that can be derivatized to include aphosphoramidate or phosphonoamidate, e.g. at the 5′ position include:

Examples of phosphoramidate or phosphonoamidate nucleoside compoundsinclude:

In one embodiment, the nucleosides that can be derivatized to include aphosphoramidate or phosphonoamidate, e.g. at the 5′ position include:

In one embodiment, phosphoramidate or phosphonoamidate nucleosidecompounds include:

In one embodiment, the nucleosides that can be derivatized to include aphosphoramidate or phosphonoamidate, e.g. at the 5′ position include:

In one embodiment, phosphoramidate or phosphonoamidate nucleosidecompounds include:

In one aspect, the compounds described herein are provided oradministered in combination with a second therapeutic agent, such as oneuseful for the treatment or prevention of HBV and/or HCV infections.Exemplary therapeutic agents are described in detail in the sectionsbelow.

In another aspect, provided are pharmaceutical compositions, single unitdosage forms, and kits suitable for use in treating or preventingdisorders such as HBV and/or HCV infections which comprise atherapeutically or prophylactically effective amount of a compounddescribed herein, e.g. of Formula I, IIa or IIb, and a therapeuticallyor prophylactically effective amount of a second therapeutic such as oneuseful for the treatment or prevention of HBV and/or HCV infections.

In certain embodiments, a method of treatment of a liver disorder isprovided comprising administering to an individual in need thereof atreatment effective amount of a phosphoramidate or phosphonoamidatederivative of a nucleoside or nucleoside analogue, wherein optionallythe derivative is a S-pivaloyl-2-thioethyl phosphoramidate orS-pivaloyl-2-thioethyl phosphonoamidate derivative. The derivative isoptionally selected from the compounds disclosed herein.

In some embodiments, provided herein are:

-   (a) compounds as described herein, e.g. of Formula I, IIa or IIb,    and pharmaceutically acceptable salts and compositions thereof;-   (b) compounds as described herein, e.g. of Formula I, IIa or IIb,    and pharmaceutically acceptable salts and compositions thereof for    use in the treatment and/or prophylaxis of a liver disorder    including Flaviviridae infection, especially in individuals    diagnosed as having a Flaviviridae infection or being at risk of    becoming infected by hepatitis C;-   (c) processes for the preparation of compounds as described herein,    e.g. of Formula I, II or IIb, as described in more detail below;-   (d) pharmaceutical formulations comprising a compound as described    herein, e.g. of Formula I, IIa or IIb, or a pharmaceutically    acceptable salt thereof together with a pharmaceutically acceptable    carrier or diluent;-   (e) pharmaceutical formulations comprising a compound as described    herein, e.g. of Formula I, IIa or IIb, or a pharmaceutically    acceptable salt thereof together with one or more other effective    anti-HCV agents, optionally in a pharmaceutically acceptable carrier    or diluent;-   (f) a method for the treatment and/or prophylaxis of a host infected    with Flaviviridae that includes the administration of an effective    amount of a compound as described herein, e.g. of Formula I, IIa or    IIb, its pharmaceutically acceptable salt or composition;-   (g) a method for the treatment and/or prophylaxis of a host infected    with Flaviviridae that includes the administration of an effective    amount of a compounds as described herein, e.g. of Formula I, IIa or    IIb, its pharmaceutically acceptable salt or composition in    combination and/or alternation with one or more effective anti-HCV    agent;-   (h) compounds as described herein, e.g. of Formula I, IIa or IIb,    and pharmaceutically acceptable salts and compositions thereof for    use in the treatment and/or prophylaxis of a HBV infection,    especially in individuals diagnosed as having an HBV infection or    being at risk of becoming infected by hepatitis B;-   (i) pharmaceutical formulations comprising a compound as described    herein, e.g. of Formula I, IIa or IIb, or a pharmaceutically    acceptable salt thereof together with one or more other effective    anti-HBV agents, optionally in a pharmaceutically acceptable carrier    or diluent;-   (j) a method for the treatment and/or prophylaxis of hepatitis B    infections and other related conditions such as anti-HBV antibody    positive and HBV-positive conditions, chronic liver inflammation    caused by HBV, cirrhosis, acute hepatitis, fulminant hepatitis,    chronic persistent hepatitis, and fatigue that includes    administering an effective amount of a compound as described herein,    e.g. of Formula I, IIa or IIb, or its pharmaceutically acceptable    salt or composition.-   (k) a prophylactic method to prevent or retard the progression of    clinical illness in individuals who are anti-HBV antibody or    HBV-antigen positive or who have been exposed to HBV.

Flaviviridae which can be treated are, e.g., discussed generally inFields Virology, Editors: Fields, B. N., Knipe, D. M., and Howley, P.M., Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 31, 1996. Ina particular embodiment of the invention, the Flaviviridae is HCV. In analternate embodiment, the Flaviviridae is a flavivirus or pestivirus.Specific flaviviruses include, without limitation: Absettarov, Alfuy,Apoi, Aroa, Bagaza, Banzi, Bouboui, Bussuquara, Cacipacore, CareyIsland, Dakar bat, Dengue 1, Dengue 2, Dengue 3, Dengue 4, Edge Hill,Entebbe bat, Gadgets Gully, Hanzalova, Hypr, Ilheus, Israel turkeymeningoencephalitis, Japanese encephalitis, Jugra, Jutiapa, Kadam,Karshi, Kedougou, Kokobera, Koutango, Kumlinge, Kunjin, Kyasanur Forestdisease, Langat, Louping ill, Meaban, Modoc, Montana myotisleukoencephalitis, Murray valley encephalitis, Naranjal, Negishi, Ntaya,Omsk hemorrhagic fever, Phnom-Penh bat, Powassan, Rio Bravo, Rocio,Royal Farm, Russian spring-summer encephalitis, Saboya, St. Louisencephalitis, Sal Vieja, San Perlita, Saumarez Reef, Sepik, Sokuluk,Spondweni, Stratford, Tembusu, Tyuleniy, Uganda S, Usutu, Wesselsbron,West Nile, Yaounde, Yellow fever, and Zika.

Pestiviruses which can be treated are discussed generally in FieldsVirology, Editors: Fields, B. N., Knipe, D. M., and Howley, P. M.,Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 33, 1996.Specific pestiviruses include, without limitation: bovine viral diarrheavirus (“BVDV”), classical swine fever virus (“CSFV,” also called hogcholera virus), and border disease virus (“BDV”).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts depletion of NM108 hydroxySATE phosphoramidate (B299)after incubation with and without NADPH in monkey liver S9.

FIG. 2 depicts depletion of NM107 hydroxySATE phosphoramidate (B102)after incubation with and without NADPH in monkey liver S9.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Provided herein are compounds, compositions and methods useful fortreating liver disorders such as HBV and/or HCV infection in a subject.Further provided are dosage forms useful for such methods.

DEFINITIONS

When referring to the compounds provided herein, the following termshave the following meanings unless indicated otherwise.

The term “alkyl”, as used herein, unless otherwise specified, includes asaturated straight, branched, or cyclic, primary, secondary, or tertiaryhydrocarbon of typically C₁ to C₁₀, and specifically includes methyl,CF₃, CCl₃, CFCl₂, CF₂Cl, ethyl, CH₂CF₃, CF₂CF₃, propyl, isopropyl,cyclopropyl, butyl, isobutyl, secbutyl, t-butyl, pentyl, cyclopentyl,isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl,3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. The termincludes both substituted and unsubstituted alkyl groups, andparticularly includes halogenated alkyl groups, and even moreparticularly fluorinated alkyl groups. Non-limiting examples of moietieswith which the alkyl group can be substituted are selected from thegroup consisting of halogen (fluoro, chloro, bromo or iodo), hydroxyl,amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonicacid, sulfate, phosphonic acid, phosphate, or phosphonate, eitherunprotected, or protected as necessary, as known to those skilled in theart, for example, as taught in Greene, et al., Protective Groups inOrganic Synthesis, John Wiley and Sons, Second Edition, 1991, herebyincorporated by reference.

The term “lower alkyl”, as used herein, and unless otherwise specified,includes a C₁ to C₄ saturated straight, branched, or if appropriate, acyclic (for example, cyclopropyl) alkyl group, including bothsubstituted and unsubstituted moieties.

“Alkylene” includes divalent saturated aliphatic hydrocarbon groupsparticularly having up to about 11 carbon atoms and more particularly 1to 6 carbon atoms which can be straight-chained or branched. This termis exemplified by groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—),the propylene isomers (e.g., —CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like.

“Alkenyl” includes monovalent olefinically unsaturated hydrocarbongroups, in certain embodiment, having up to about 11 carbon atoms, from2 to 8 carbon atoms, or from 2 to 6 carbon atoms, which can bestraight-chained or branched and having at least 1 or from 1 to 2 sitesof olefinic unsaturation. Exemplary alkenyl groups include ethenyl(—CH═CH₂), n-propenyl (—CH₂CH═CH₂), isopropenyl (—C(CH₃)═CH₂), vinyl andsubstituted vinyl, and the like.

“Alkenylene” includes divalent olefinically unsaturated hydrocarbongroups, in certain embodiments, having up to about 11 carbon atoms orfrom 2 to 6 carbon atoms which can be straight-chained or branched andhaving at least 1 or from 1 to 2 sites of olefinic unsaturation. Thisterm is exemplified by groups such as ethenylene (—CH═CH—), thepropenylene isomers (e.g., —CH═CHCH₂— and —C(CH₃)═CH— and —CH═C(CH₃)—)and the like.

“Alkynyl” includes acetylenically unsaturated hydrocarbon groups, incertain embodiments, having up to about 11 carbon atoms or from 2 to 6carbon atoms which can be straight-chained or branched and having atleast 1 or from 1 to 2 sites of alkynyl unsaturation. Non-limitingexamples of alkynyl groups include acetylenic, ethynyl (—C≡CH),propargyl (—CH₂C≡CH), and the like.

The term “aryl”, as used herein, and unless otherwise specified,includes phenyl, biphenyl, or naphthyl, and preferably phenyl. The termincludes both substituted and unsubstituted moieties. The aryl group canbe substituted with any described moiety, including, but not limited to,one or more moieties selected from the group consisting of halogen(fluoro, chloro, bromo or iodo), alkyl, haloalkyl, hydroxyl, amino,alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid,sulfate, phosphoric acid, phosphate, or phosphonate, either unprotected,or protected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons, Second Edition, 1991.

“Alkoxy” includes the group —OR where R is alkyl. Particular alkoxygroups include, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy,1,2-dimethylbutoxy, and the like.

“Alkoxycarbonyl” includes a radical —C(O)-alkoxy where alkoxy is asdefined herein.

“Amino” includes the radical —NH₂.

“Carboxyl” includes the radical —C(O)OH.

The term “alkylamino” or “arylamino” includes an amino group that hasone or two alkyl or aryl substituents, respectively. Unless otherwisespecifically stated in this application, when alkyl is a suitablemoiety, lower alkyl is preferred. Similarly, when alkyl or lower alkylis a suitable moiety, unsubstituted alkyl or lower alkyl is preferred.

“Halogen” or “halo” includes chloro, bromo, fluoro or iodo.

“Monoalkylamino” includes the group alkyl-NR′—, wherein R′ is selectedfrom hydrogen and alkyl.

“Thioalkoxy” includes the group —SR where R is alkyl.

The term “protected” as used herein and unless otherwise defined refersto a group that is added to an oxygen, nitrogen, or phosphorus atom toprevent its further reaction or for other purposes. A wide variety ofoxygen and nitrogen protecting groups are known to those skilled in theart of organic synthesis.

“Pharmaceutically acceptable salt” includes any salt of a compoundprovided herein which retains its biological properties and which is nottoxic or otherwise undesirable for pharmaceutical use. Such salts may bederived from a variety of organic and inorganic counter-ions well knownin the art. Such salts include: (1) acid addition salts formed withorganic or inorganic acids such as hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, sulfamic, acetic, trifluoroacetic, trichloroacetic,propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic,lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric,tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, picric,cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic,1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic,4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic,camphoric, camphorsulfonic,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic,3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric,gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic,cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2)salts formed when an acidic proton present in the parent compound either(a) is replaced by a metal ion, e.g., an alkali metal ion, an alkalineearth ion or an aluminum ion, or alkali metal or alkaline earth metalhydroxides, such as sodium, potassium, calcium, magnesium, aluminum,lithium, zinc, and barium hydroxide, ammonia or (b) coordinates with anorganic base, such as aliphatic, alicyclic, or aromatic organic amines,such as ammonia, methylamine, dimethylamine, diethylamine, picoline,ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethylene-diamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane,tetramethylammonium hydroxide, and the like.

Salts further include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium and the like, and whenthe compound contains a basic functionality, salts of non-toxic organicor inorganic acids, such as hydrohalides, e.g. hydrochloride andhydrobromide, sulfate, phosphate, sulfamate, nitrate, acetate,trifluoroacetate, trichloroacetate, propionate, hexanoate,cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate,malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate,tartarate, citrate, benzoate, 3-(4-hydroxybenzoyl)benzoate, picrate,cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate),ethanesulfonate, 1,2-ethane-disulfonate, 2-hydroxyethanesulfonate,benzenesulfonate (besylate), 4-chlorobenzenesulfonate,2-naphthalenesulfonate, 4-toluenesulfonate, camphorate,camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate,glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate,lauryl sulfate, gluconate, benzoate, glutamate, hydroxynaphthoate,salicylate, stearate, cyclohexylsulfamate, quinate, muconate and thelike.

The term “alkaryl” or “alkylaryl” includes an aryl group with an alkylsubstituent. The term aralkyl or arylalkyl includes an alkyl group withan aryl substituent.

The term “purine” or “pyrimidine” base includes, but is not limited to,adenine, N⁶-alkylpurines, N⁶-acylpurines (wherein acyl is C(O)(alkyl,aryl, alkylaryl, or arylalkyl), N⁶-benzylpurine, N⁶-halopurine,N⁶-vinylpurine, N⁶-acetylenic purine, N⁶-acyl purine, N⁶-hydroxyalkylpurine, N⁶-alkylaminopurine, N⁶-thioalkyl purine, N²-alkylpurines,N²-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine,5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or4-mercaptopyrmidine, uracil, 5-halouracil, including 5-fluorouracil,C⁵-alkylpyrimidines, C⁵-benzylpyrimidines, C⁵-halopyrimidines,C⁵-vinylpyrimidine, C⁵-acetylenic pyrimidine, C⁵-acyl pyrimidine,C⁵-hydroxyalkyl purine, C⁵-amidopyrimidine, C⁵-cyanopyrimidine,C⁵-iodopyrimidine, C⁶-iodo-pyrimidine, C⁵—Br-vinyl pyrimidine,C⁶—Br-vinyl pyrimidine, C⁵-nitropyrimidine, C⁵-amino-pyrimidine,N²-alkylpurines, N²-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl,triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, andpyrazolopyrimidinyl. Purine bases include, but are not limited to,guanine, adenine, hypoxanthine, 7-deazaguanine, 7-deazaadenine,2,6-diaminopurine, and 6-chloropurine. Functional oxygen and nitrogengroups on the base can be protected as necessary or desired. Suitableprotecting groups are well known to those skilled in the art, andinclude trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, andt-butyldiphenylsilyl, trityl, alkyl groups, and acyl groups such asacetyl and propionyl, methanesulfonyl, and p-toluenesulfonyl.

The term “acyl” or “O-linked ester” includes a group of the formulaC(O)R′, wherein R′ is an straight, branched, or cyclic alkyl (includinglower alkyl), carboxylate reside of amino acid, aryl including phenyl,alkaryl, arylalkyl including benzyl, alkoxyalkyl includingmethoxymethyl, aryloxyalkyl such as phenoxymethyl; or substituted alkyl(including lower alkyl), aryl including phenyl optionally substitutedwith chloro, bromo, fluoro, iodo, C₁ to C₄ alkyl or C₁ to C₄ alkoxy,sulfonate esters such as alkyl or arylalkyl sulphonyl includingmethanesulfonyl, the mono, di or triphosphate ester, trityl ormonomethoxy-trityl, substituted benzyl, alkaryl, arylalkyl includingbenzyl, alkoxyalkyl including methoxymethyl, aryloxyalkyl such asphenoxymethyl. Aryl groups in the esters optimally comprise a phenylgroup. In particular, acyl groups include acetyl, trifluoroacetyl,methylacetyl, cyclopropylacetyl, propionyl, butyryl, hexanoyl,heptanoyl, octanoyl, neo-heptanoyl, phenylacetyl,2-acetoxy-2-phenylacetyl, diphenylacetyl,α-methoxy-α-trifluoromethyl-phenylacetyl, bromoacetyl,2-nitro-benzeneacetyl, 4-chloro-benzeneacetyl,2-chloro-2,2-diphenylacetyl, 2-chloro-2-phenylacetyl, trimethylacetyl,chlorodifluoroacetyl, perfluoroacetyl, fluoroacetyl,bromodifluoroacetyl, methoxyacetyl, 2-thiopheneacetyl,chlorosulfonylacetyl, 3-methoxyphenylacetyl, phenoxyacetyl,tert-butylacetyl, trichloroacetyl, monochloro-acetyl, dichloroacetyl,7H-dodecafluoro-heptanoyl, perfluoro-heptanoyl,7H-dodeca-fluoroheptanoyl, 7-chlorododecafluoro-heptanoyl,7-chloro-dodecafluoro-heptanoyl, 7H-dodecafluoroheptanoyl,7H-dodeca-fluoroheptanoyl, nona-fluoro-3,6-dioxa-heptanoyl,nonafluoro-3,6-dioxaheptanoyl, perfluoroheptanoyl, methoxybenzoyl,methyl 3-amino-5-phenylthiophene-2-carboxyl,3,6-dichloro-2-methoxy-benzoyl, 4-(1,1,2,2-tetrafluoro-ethoxy)-benzoyl,2-bromo-propionyl, omega-aminocapryl, decanoyl, n-pentadecanoyl,stearyl, 3-cyclopentyl-propionyl, 1-benzene-carboxyl, O-acetylmandelyl,pivaloyl acetyl, 1-adamantane-carboxyl, cyclohexane-carboxyl,2,6-pyridinedicarboxyl, cyclopropane-carboxyl, cyclobutane-carboxyl,perfluorocyclohexyl carboxyl, 4-methylbenzoyl, chloromethyl isoxazolylcarbonyl, perfluorocyclohexyl carboxyl, crotonyl,1-methyl-1H-indazole-3-carbonyl, 2-propenyl, isovaleryl,1-pyrrolidinecarbonyl, 4-phenylbenzoyl.

The term “amino acid” includes naturally occurring and synthetic α, β γor δ amino acids, and includes but is not limited to, amino acids foundin proteins, i.e. glycine, alanine, valine, leucine, isoleucine,methionine, phenylalanine, tryptophan, proline, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,arginine and histidine. In a preferred embodiment, the amino acid is inthe L-configuration. Alternatively, the amino acid can be a derivativeof alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl, phenylalaninyl,tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl,tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl,argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleuccinyl,β-prolinyl, β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl,β-serinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl,β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl orβ-histidinyl.

As used herein, the term “substantially free of” or “substantially inthe absence of” with respect to a nucleoside composition includes anucleoside composition that includes at least 85 or 90% by weight,preferably 95%, 98%, 99% or 100% by weight, of the designated enantiomerof that nucleoside. In a preferred embodiment, in the methods andcompounds of this invention, the compounds are substantially free ofenantiomers.

Similarly, the term “isolated” with respect to a nucleoside compositionincludes a nucleoside composition that includes at least 85, 90%, 95%,98%, 99% to 100% by weight, of the nucleoside, the remainder comprisingother chemical species or enantiomers.

“Solvate” includes a compound provided herein or a salt thereof, thatfurther includes a stoichiometric or non-stoichiometric amount ofsolvent bound by non-covalent intermolecular forces. Where the solventis water, the solvate is a hydrate.

The term “host”, as used herein, includes any unicellular ormulticellular organism in which the virus can replicate, including celllines and animals, and preferably a human. Alternatively, the host canbe carrying a part of the Flaviviridae viral genome, whose replicationor function can be altered by the compounds of the present invention.The term host specifically includes infected cells, cells transfectedwith all or part of the Flaviviridae genome and animals, in particular,primates (including chimpanzees) and humans. In most animal applicationsof the present invention, the host is a human patient. Veterinaryapplications, in certain indications, however, are clearly anticipatedby the present invention (such as chimpanzees).

As used herein, the terms “subject” and “patient” are usedinterchangeably herein. The terms “subject” and “subjects” refer to ananimal, such as a mammal including a non-primate (e.g., a cow, pig,horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey such as acynomolgous monkey, a chimpanzee and a human), and for example, a human.In one embodiment, the subject is refractory or non-responsive tocurrent treatments for hepatitis C infection. In another embodiment, thesubject is a farm animal (e.g., a horse, a cow, a pig, etc.) or a pet(e.g., a dog or a cat). In one embodiment, the subject is a human.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to any agent(s) which can be used in the treatment or preventionof a disorder or one or more symptoms thereof. In certain embodiments,the term “therapeutic agent” includes a compound provided herein. In oneembodiment, a therapeutic agent is an agent which is known to be usefulfor, or has been or is currently being used for the treatment orprevention of a disorder or one or more symptoms thereof.

“Therapeutically effective amount” includes an amount of a compound orcomposition that, when administered to a subject for treating a disease,is sufficient to effect such treatment for the disease. A“therapeutically effective amount” can vary depending on, inter alia,the compound, the disease and its severity, and the age, weight, etc.,of the subject to be treated.

“Treating” or “treatment” of any disease or disorder refers, in oneembodiment, to ameliorating a disease or disorder that exists in asubject. In another embodiment, “treating” or “treatment” includesameliorating at least one physical parameter, which may be indiscernibleby the subject. In yet another embodiment, “treating” or “treatment”includes modulating the disease or disorder, either physically (e.g.,stabilization of a discernible symptom) or physiologically (e.g.,stabilization of a physical parameter) or both. In yet anotherembodiment, “treating” or “treatment” includes delaying the onset of thedisease or disorder.

As used herein, the terms “prophylactic agent” and “prophylactic agents”as used refer to any agent(s) which can be used in the prevention of adisorder or one or more symptoms thereof. In certain embodiments, theterm “prophylactic agent” includes a compound provided herein. Incertain other embodiments, the term “prophylactic agent” does not refera compound provided herein. For example, a prophylactic agent is anagent which is known to be useful for, or has been or is currently beingused to the prevent or impede the onset, development, progression and/orseverity of a disorder.

As used herein, the phrase “prophylactically effective amount” includesthe amount of a therapy (e.g., prophylactic agent) which is sufficientto result in the prevention or reduction of the development, recurrenceor onset of one or more symptoms associated with a disorder (, or toenhance or improve the prophylactic effect(s) of another therapy (e.g.,another prophylactic agent).

Compounds

Phosphoramidate and phosphonoamidate compounds of a variety oftherapeutic agents can be formed using methods available in the art andthose disclosed herein. Such compounds can be used in some embodimentsto enhance delivery of the drug to the liver. In one embodiment, thecompound is an S-acyl-2-thioethyl phosphoramidate or anS-acyl-2-thioethyl phosphonoamidate derivative, e.g., anS-pivaloyl-2-thioethyl phosphoroamidate, anS-hydroxypivaloyl-2-thioethyl phosphoroamidate, anS-pivaloyl-2-thioethyl phosphonoamidate or anS-hydroxypivaloyl-2-thioethyl phosphonoamidate. Therapeutic agents thatcan be derivatized to compound form include an anti-viral agent thatincludes, or has been derivatized to include a reactive group forattachment of the phosphoramidate or phosphonoamidate moiety, includingbut not limited to nucleosides and nucleoside analogues includingacyclic nucleosides. Therapeutic agents that can be derivatized tocompound form also include an anti-viral agent that includes, or hasbeen derivatized to include a phosphate or phorphonate group that can bederivatized to form a phosphoramidate or phosphonoamidate moiety,including but not limited to nucleosides and nucleoside analoguesincluding acyclic nucleosides.

Nucleosides that can be derivatized include any R¹ as disclosed herein.Examples of nucleosides that can be derivatized to include aphosphoramidate or phosphonoamidate, e.g. at the 5′, 3′ or 2′ positioninclude:

Examples of phosphoramidate or phosphonoamidate nucleoside compoundsinclude:

Phosphoramidate or phosphonoamidate compounds of other nucleosides andnucleoside analogues described herein and known in the art can be formedas described herein and used for the treatment of liver disorders. Thephosphoramidate or phosphonoamidate moiety can be e.g., at the 5′position.

In one embodiment, provided herein are compounds, as well as saltsthereof, and compositions comprising the compounds, that are useful fortreatment of disorders of the liver, including HBV and/or HCVinfections. In one embodiment, the phosphoramidate or phosphonoamidatecompound provided herein is a compound of Formula IIa or IIb:

or a pharmaceutically acceptable salt, solvate, a stereoisomeric,tautomeric or polymorphic form thereof, wherein;

R^(y) is alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl,cycloalkenyl, amino, heterocyclyl or heteroaryl, all optionallysubstituted;

R^(a) and R^(b) are selected as follows:

i) R^(a) and R^(b) are each independently hydrogen, alkyl, carboxyalkyl,hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl,alkoxycarbonylalkyl, aryl, arylalkyl, cycloalkyl, heteroaryl orheterocyclyl, all optionally substituted; or

ii) R^(a) and R^(b) together with the nitrogen atom on which they aresubstituted form a 3-7 membered heterocyclic or heteroaryl ring; and

R¹ is an anti-viral drug (which includes a moiety derivable by removalof a hydrogen from a hydroxy group of an anti-viral drug).

In certain embodiments, the compound of Formula IIa or IIb is selectedwith a proviso that when R^(y) is tert-butyl or hydroxy-tert-butyl, thenR¹ is not 3′-azido-2′,3′-dideoxythymidine.

In certain embodiments, R¹, R^(a), R^(b) and R^(y) are optionallysubstituted with one or more substituents as defined in the definitions.

In certain embodiments, the compounds are of Formula IIa or IIb, whereinR^(y) is alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl,cycloalkenyl, amino, heterocyclyl or heteroaryl;

R^(a) and R^(b) are each independently hydrogen, alkyl, carboxyalkyl,hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl,alkoxycarbonylalkyl, aryl, arylalkyl, cycloalkyl, heteroaryl orheterocyclyl, all optionally substituted; and

R¹ is an anti-viral drug (which is meant to include a moiety derivableby removal of a hydrogen from a hydroxy group of an anti-viral drug).

In one embodiment, R¹ is a nucleoside comprising a cyclic or acyclicsugar or analog thereof, including any nucleoside or analogue thereofdescribed herein or known in the art.

Exemplary nucleoside drugs useful in the treatment of hepatitis Cinfection that can be derivatized as described herein are:

Name Structure Ribavirin

Viramidine

Valopicitabine (NM283)

2′-C-methylcytidine (NM107)

PSI-6130

MK-0608

7-Fluoro-MK-0608

NM108

Exemplary non-nucleoside drugs that can be derivatized as describedherein are:

Name Structure Resiquimod

Celgosivir

Gliotoxin

Exemplary nucleoside drugs useful in the treatment of hepatitis Binfection that can be derivatized as described herein are:

Name Structure Lamivudine or 3TC or Epivir ®

Entecavir

Telbivudine or L-dT

Racivir

Emtricitabine or (−)FTC

Clevudine or L-FMAU

Amdoxovir

Valtorcitabine

Torcitabine (L-dC)

Tenofovir or PMPA

Adefovir or PMEA

L-cytidine

A phosphoramidate or phosphonoamidate compound of acyclovir, L-ddA orD-ddA can be administered for treatment of Hepatitis B, an example ofwhich is shown below:

Where the nucleoside analog already includes a phosphonate, thatphosphonate group can be incorporated in the phosphonoamidate moietyshown in the formulas herein, as shown by way of example in thephosphonoamidate of adefovir:

Thus, in certain embodiments of the compounds of Formula II below:

-   -   the moiety:

is derived from a drug that is an acyclic nucleoside phosphonate, i.e.:

which is e.g. PMEA (9-[2-(phosphonomethoxy)ethyl]adenine (adefovir).

In certain embodiments according to Formula IIa or IIb, R^(y) issubstituted alkyl, e.g. hydroxyalkyl or aminoalkyl; and R^(a) and R^(b)are independently hydrogen, alkyl, substituted alkyl, benzyl orsubstituted benzyl, for instance hydroxy- or amino-substituted alkyl orbenzyl. In another embodiment, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c)where each R^(c) is independently alkyl, substituted alkyl, aryl orsubstituted aryl, for instance hydroxy- or amino-substituted alkyl oraryl; and R^(a) and R^(b) are independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl, for instance hydroxy- oramino-substituted alkyl or benzyl.

In a further embodiment, R^(a) and R^(b) are independently benzyl orsubstituted alkyl. In a further embodiment, R^(y) is selected from thegroup consisting of alkyl and hydroxyalkyl. In certain embodiments,R^(y) is —C(CH₃)₂CH₂OH. In certain embodiments according to thisparagraph, R² and R³ are each hydrogen, R^(a) is hydrogen, R^(b) is—CH₂—C₆H₅ and R^(y) is —C(CH₃)₂CH₂OH.

In one embodiment, R^(y) is alkyl or hydroxyalkyl. In one embodiment,R^(y) is methyl, tert-butyl, hydroxy-tert-butyl or hydroxyethyl. Incertain embodiments, R^(y) is —C(CH₃)₂CH₂OH.

In one embodiment, R^(a) and R^(b) are each independently hydrogen,alkyl, carboxyalkyl, hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl,aminocarbonylalkyl, alkoxycarbonylalkyl, aryl or arylalkyl, wherein thealkyl groups can be further substituted with one or more substitutents.In one embodiment, at least one of R^(a) or R^(b) is other thanhydrogen. In one embodiment, R^(a) and R^(b) are each independentlyhydrogen, methyl or benzyl.

In certain embodiments, R^(y) is —C(CH₃)₂CH₂OH and R^(a) and R^(b) areeach independently hydrogen, methyl or benzyl. In certain embodiments,R^(y) is —C(CH₃)₂CH₂OH and R^(a) is hydrogen and R^(b) is benzyl.

In another embodiment, the compound provided herein is a compound offormula:

wherein R¹ and R^(y) are as defined in Formula IIa or IIb.

In certain embodiments of Formula IIIa, b, c or d:

R^(y) is substituted alkyl, e.g., hydroxyalkyl or aminoalkyl; and

In another embodiment, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c) whereeach R^(c) is independently alkyl, substituted alkyl, aryl orsubstituted aryl, for instance hydroxy- or amino-substituted alkyl oraryl; and R^(a) and R^(b) are independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl, for instance hydroxy- oramino-substituted alkyl or hydroxy-, amino-, alkyl-, haloalkyl- ortrifluoromethyl-substituted benzyl. In certain embodiments, R^(a) andR^(b) together with the nitrogen atom on which they are substituted forma 3-7 membered heterocyclic or heteroaryl ring.

In one embodiment, R^(y) is alkyl or hydroxyalkyl. In one embodiment,R^(y) is methyl, tert-butyl, hydroxy-tert-butyl or hydroxyethyl. In oneembodiment, R^(y) is —C(CH₃)₂CH₂OH.

In certain embodiments according to Formula IIIa or IIIb, R^(y) issubstituted alkyl, e.g. hydroxyalkyl or aminoalkyl. In anotherembodiment, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c) where each R^(c) isindependently alkyl, substituted alkyl, aryl or substituted aryl, forinstance hydroxy- or amino-substituted alkyl or aryl. In a furtherembodiment, R^(y) is selected from the group consisting of alkyl andhydroxyalkyl. In certain embodiments, R^(y) is —C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

wherein R¹, R^(a) and R^(b) are e.g. as defined in Formula IIa or IIb).

In certain embodiments of Formula IVa or IVb:

R¹ is an antiviral drug, such as a nucleoside or nucleoside derivative;and

R^(a) and R^(b) are each independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl, for instance hydroxy- oramino-substituted alkyl or hydroxy-, amino-, alkyl-, haloalkyl- ortrifluoromethyl-substituted benzyl. In a further embodiment, R^(a) andR^(b) are independently H, benzyl or substituted alkyl. In certainembodiments, R^(a) and R^(b) together with the nitrogen atom on whichthey are substituted form a 3-7 membered heterocyclic or heteroarylring.

In certain embodiments of Formula IVa or IVb:

R¹ is an antiviral drug, such as a nucleoside or nucleoside derivative;and

R^(a) and R^(b) are each independently hydrogen, alkyl, carboxyalkyl,hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl,alkoxycarbonylalkyl, aryl, arylalkyl, cycloalkyl, heteroaryl, orheterocyclyl, all optionally substituted; and

wherein, in one embodiment, one of R^(a) and R^(b) is H and the other isalkyl optionally substituted with aryl, benzyl, or heteroaryl, eachoptionally substituted.

In another embodiment, the compound provided herein is a compound offormula:

wherein R¹ is as defined in Formula IIa or

In certain embodiments, R¹ is an anti-viral nucleoside analog useful fortreatment of HCV virus infection selected from ribavirin, viramidine,2′-C-methylcytidine, 2′-C-methylguanosine, valopicitabine (NM 283),MK-0608 and PSI-6130. As used herein, where R¹ is an analogue of anucleoside, such as acyclovir, that itself includes a phosphonate group,that phosphonate can be in the form of the phosphonoamidate in theformulas disclosed herein. Thus, e.g., in formula Va or Vb, theR¹P(O)O-fragment is derived from the nucleoside analog that includes aphosphonate.

In certain embodiments, R¹ is an anti-viral nucleoside analog useful fortreatment of HBV virus infection selected from lamivudine (Epivir-HBV,Zeffix, or Heptodin), adefovir, entecavir (Baraclude), telbivudine(Tyzeka, Sebivo), emtricitabine (FTC), clevudine (L-FMAU), viread(Tenofovir), torcitabine, valtorcitabine (monoval LdC), amdoxovir (DAPD)and RCV (Racivir).

Further exemplary anti-viral nucleoside analogs that can be used as R¹are disclosed in International Publication Nos. WO2005021568;WO2006094347 and WO2006093987 and US Patent Publication No.US20050215510.

In certain embodiments, R¹ is a non-nucleoside anti-viral useful fortreatment of HBV virus infection selected from resiquimod or celgosivir.

In one embodiment, R¹ is an immunosuppressant, such as combretastatinA-4, mycophenolic acid, pentostatin, nelarabine or mitoxantrone.

In one embodiment, R¹ is an interfering RNA (iRNA) based antivirals,including short interfering RNA (siRNA) based antivirals. Such compoundsare described in International Patent Publication Nos. WO/03/070750 andWO 2005/012525, U.S. Pat. Nos. 7,176,304; 7,109,165; 7,041,817;7,034,009; 7,022,828; 6,852,535 and 6,849,726 and US Patent PublicationNo. US 2004/0209831.

In another embodiment, the compound provided herein is a compound offormula:

wherein R^(a), R^(b) and R^(y) are as defined in Formula IIa or IIb andR² and R³ are each independently H; straight chained, branched or cyclicalkyl; acyl (including lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl,CO-aryloxyalkyl, CO-substituted aryl, sulfonate ester such as alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted; alkylsulfonyl, arylsulfonyl,aralkylsulfonyl, a lipid, such as a phospholipid; an amino acid; andamino acid residue, a carbohydrate; a peptide; cholesterol; or otherpharmaceutically acceptable leaving group which is capable of providinga compound wherein R² and/or R³ is independently H or phosphate(including mono-, di- or triphosphate), for example when administered invivo; or R² and R³ are linked to form a cyclic group by an alkyl, esteror carbamate linkage. Each R^(L) is independently H, carbamyl, straightchained, branched or cyclic alkyl; acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester such as alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted; alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, a lipid,such as a phospholipid; an amino acid; an amino acid residue; or acarbohydrate. In certain embodiments, R² and R³ are each hydrogen, R^(a)is hydrogen, R^(b) is —CH₂—C₆H₅ and R^(y) is —C(CH₃)₂CH₂OH. In certainembodiments according to this paragraph, R² and R³ are each H; R^(y) issubstituted alkyl, e.g. hydroxyalkyl or aminoalkyl; and R^(a) and R^(b)are independently hydrogen, alkyl, substituted alkyl, benzyl orsubstituted benzyl, for instance hydroxy- or amino-substituted alkyl orbenzyl. In another embodiment, R² and R³ are each H; R^(y) is —OR^(c),—C(R^(c))₃ or —NHR^(c) where each R^(c) is independently alkyl,substituted alkyl, aryl or substituted aryl, for instance hydroxy- oramino-substituted alkyl or aryl; and R^(a) and R^(b) are independentlyhydrogen, alkyl, substituted alkyl, benzyl or substituted benzyl, forinstance hydroxy- or amino-substituted alkyl or benzyl. In a furtherembodiment, R² and R³ are each H; R^(a) and R^(b) are independentlybenzyl or substituted alkyl. In a further embodiment, R² and R³ are eachH; R^(y) is selected from the group consisting of alkyl andhydroxyalkyl. In certain embodiments, R² and R³ are each H; R^(y) is—C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

wherein R^(a), R^(b) and R^(y) are as defined in Formula IIa or IIb andR^(e) is hydrogen or alkyl. Each R^(L) is independently H, carbamyl,straight chained, branched or cyclic alkyl; acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester such as alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, suchas a phospholipid; an amino acid; an amino acid residue; or acarbohydrate. In certain embodiments, R^(e) is methyl, ethyl or propyl,R^(a) is hydrogen, R^(b) is —CH₂—C₆H₅ and R^(y) is —C(CH₃)₂CH₂OH. Incertain embodiments according to this paragraph, R² and R³ are each H;R^(y) is substituted alkyl, e.g. hydroxyalkyl or aminoalkyl; and R^(a)and R^(b) are independently hydrogen, alkyl, substituted alkyl, benzylor substituted benzyl, for instance hydroxy- or amino-substituted alkylor benzyl. In another embodiment, R^(e) is methyl, ethyl or propyl; R²and R³ are each H; R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c) where eachR^(c) is independently alkyl, substituted alkyl, aryl or substitutedaryl, for instance hydroxy- or amino-substituted alkyl or aryl; andR^(a) and R^(b) are independently hydrogen, alkyl, substituted alkyl,benzyl or substituted benzyl, for instance hydroxy- or amino-substitutedalkyl or benzyl. In a further embodiment, R^(e) is methyl, ethyl orpropyl; R² and R³ are each H; R^(a) and R^(b) are independently benzylor substituted alkyl. In a further embodiment, R² and R³ are each H;R^(y) is selected from the group consisting of alkyl and hydroxyalkyl.In certain embodiments, R^(e) is methyl, ethyl or propyl; R² and R³ areeach H; R^(y) is —C(CH₃)₂CH₂OH. In certain embodiments according to thisparagraph, R¹ is chosen from nucleosides described in U.S. PatentApplication Publication No. US 2006/0111324 A1, the content of which ishereby incorporated by reference in its entirety.

In another embodiment, the compound provided herein is a compound offormula:

wherein R^(a), R^(b) and R^(y) are as defined in Formula IIa or IIb.Each R^(L) is independently H, carbamyl, straight chained, branched orcyclic alkyl; acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate estersuch as alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted;alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, a lipid, such as aphospholipid; an amino acid; an amino acid residue; or a carbohydrate.In certain embodiments according to this paragraph, each R^(L) ishydrogen, R^(y) is substituted alkyl, e.g. hydroxyalkyl or aminoalkyl;and R^(a) and R^(b) are independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl, for instance hydroxy- oramino-substituted alkyl or benzyl. In another embodiment, each R^(L) ishydrogen, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c) where each R^(c) isindependently alkyl, substituted alkyl, aryl or substituted aryl, forinstance hydroxy- or amino-substituted alkyl or aryl; and R^(a) andR^(b) are independently hydrogen, alkyl, substituted alkyl, benzyl orsubstituted benzyl, for instance hydroxy- or amino-substituted alkyl orbenzyl. In a further embodiment, each R^(L) is hydrogen, R^(a) and R^(b)are independently benzyl or substituted alkyl. In a further embodiment,R^(y) is selected from the group consisting of alkyl and hydroxyalkyl.In certain embodiments, each R^(L) is hydrogen and R^(y) is—C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

wherein R^(a), R^(b) and R^(y) are as defined in Formula IIa or IIb.Each R^(L) is independently H, carbamyl, straight chained, branched orcyclic alkyl; acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate estersuch as alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted;alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, a lipid, such as aphospholipid; an amino acid; an amino acid residue; or a carbohydrate.In certain embodiments according to this paragraph, each R^(L) ishydrogen, R^(y) is substituted alkyl, e.g. hydroxyalkyl or aminoalkyl;and R^(a) and R^(b) are independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl, for instance hydroxy- oramino-substituted alkyl or benzyl. In another embodiment, each R^(L) ishydrogen, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c) where each R^(c) isindependently alkyl, substituted alkyl, aryl or substituted aryl, forinstance hydroxy- or amino-substituted alkyl or aryl; and R^(a) andR^(b) are independently hydrogen, alkyl, substituted alkyl, benzyl orsubstituted benzyl, for instance hydroxy- or amino-substituted alkyl orbenzyl. In a further embodiment, each R^(L) is hydrogen, R^(a) and R^(b)are independently benzyl or substituted alkyl. In a further embodiment,R^(y) is selected from the group consisting of alkyl and hydroxyalkyl.In certain embodiments, each R^(L) is hydrogen and R^(y) is—C(CH₃)₂CH₂OH. In another embodiment, the compound provided herein is acompound of formula:

wherein R^(a), R^(b) and R^(y) are as defined in Formula IIa or IIb.Each R^(L) is independently H, carbamyl, straight chained, branched orcyclic alkyl; acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate estersuch as alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted;alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, a lipid, such as aphospholipid; an amino acid; an amino acid residue; or a carbohydrate.In certain embodiments according to this paragraph, each R^(L) ishydrogen, R^(y) is substituted alkyl, e.g. hydroxyalkyl or aminoalkyl;and R^(a) and R^(b) are independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl, for instance hydroxy- oramino-substituted alkyl or benzyl. In another embodiment, each R^(L) ishydrogen, R^(y) is —OC^(c), —C(R^(c))₃ or —NHR^(c) where each R^(c) isindependently alkyl, substituted alkyl, aryl or substituted aryl, forinstance hydroxy- or amino-substituted alkyl or aryl; and R^(a) andR^(b) are independently hydrogen, alkyl, substituted alkyl, benzyl orsubstituted benzyl, for instance hydroxy- or amino-substituted alkyl orbenzyl. In a further embodiment, each R^(L) is hydrogen, R^(a) and R^(b)are independently benzyl or substituted alkyl. In a further embodiment,R^(y) is selected from the group consisting of alkyl and hydroxyalkyl.In certain embodiments, each R^(L) is hydrogen and R^(y) is—C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

wherein R^(a), R^(b) and R^(y) are as defined in Formula IIa or IIb.Each R^(L) is independently H, carbamyl, straight chained, branched orcyclic alkyl; acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate estersuch as alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted;alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, a lipid, such as aphospholipid; an amino acid; an amino acid residue; or a carbohydrate.In certain embodiments according to this paragraph, each R^(L) ishydrogen, R^(y) is substituted alkyl, e.g. hydroxyalkyl or aminoalkyl;and R^(a) and R^(b) are independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl, for instance hydroxy- oramino-substituted alkyl or benzyl. In another embodiment, each R^(L) ishydrogen, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c) where each R^(c) isindependently alkyl, substituted alkyl, aryl or substituted aryl, forinstance hydroxy- or amino-substituted alkyl or aryl; and R^(a) andR^(b) are independently hydrogen, alkyl, substituted alkyl, benzyl orsubstituted benzyl, for instance hydroxy- or amino-substituted alkyl orbenzyl. In a further embodiment, each R^(L) is hydrogen, R^(a) and R^(b)are independently benzyl or substituted alkyl. In a further embodiment,R^(y) is selected from the group consisting of alkyl and hydroxyalkyl.In certain embodiments, each R^(L) is hydrogen and R^(y) is—C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

wherein R^(a), R^(b) and R^(y) are as defined in Formula IIa or IIb.Each R^(L) is independently H, carbamyl, straight chained, branched orcyclic alkyl; acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate estersuch as alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted;alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, a lipid, such as aphospholipid; an amino acid; an amino acid residue; or a carbohydrate.In certain embodiments according to this paragraph, each R^(L) ishydrogen, R^(y) is substituted alkyl, e.g. hydroxyalkyl or aminoalkyl;and R^(a) and R^(b) are independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl, for instance hydroxy- oramino-substituted alkyl or benzyl. In another embodiment, each R^(L) ishydrogen, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c) where each R^(c) isindependently alkyl, substituted alkyl, aryl or substituted aryl, forinstance hydroxy- or amino-substituted alkyl or aryl; and R^(a) andR^(b) are independently hydrogen, alkyl, substituted alkyl, benzyl orsubstituted benzyl, for instance hydroxy- or amino-substituted alkyl orbenzyl. In a further embodiment, each R^(L) is hydrogen, R^(a) and R^(b)are independently benzyl or substituted alkyl. In a further embodiment,R^(y) is selected from the group consisting of alkyl and hydroxyalkyl.In certain embodiments, each R^(L) is hydrogen and R^(y) is—C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

wherein R^(a), R^(b) and R^(y) are as defined in Formula IIa or IIIb.Each R^(L) is independently H, carbamyl, straight chained, branched orcyclic alkyl; acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate estersuch as alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted;alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, a lipid, such as aphospholipid; an amino acid; an amino acid residue; or a carbohydrate.In certain embodiments according to this paragraph, each R^(L) ishydrogen, R^(y) is substituted alkyl, e.g. hydroxyalkyl or aminoalkyl;and R^(a) and R^(b) are independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl, for instance hydroxy- oramino-substituted alkyl or benzyl. In another embodiment, each R^(L) ishydrogen, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c) where each R^(c) isindependently alkyl, substituted alkyl, aryl or substituted aryl, forinstance hydroxy- or amino-substituted alkyl or aryl; and R^(a) andR^(b) are independently hydrogen, alkyl, substituted alkyl, benzyl orsubstituted benzyl, for instance hydroxy- or amino-substituted alkyl orbenzyl. In a further embodiment, each R^(L) is hydrogen, R^(a) and R^(b)are independently benzyl or substituted alkyl. In a further embodiment,R^(y) is selected from the group consisting of alkyl and hydroxyalkyl.In certain embodiments, each R^(L) is hydrogen and R^(y) is—C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

wherein R^(a), R^(b) and R^(y) are as defined in Formula IIa or IIb.Each R^(L) is independently H, carbamyl, straight chained, branched orcyclic alkyl; acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate estersuch as alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted;alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, a lipid, such as aphospholipid; an amino acid; an amino acid residue; or a carbohydrate.In certain embodiments according to this paragraph, each R^(L) ishydrogen, R^(y) is substituted alkyl, e.g. hydroxyalkyl or aminoalkyl;and R^(a) and R^(b) are independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl, for instance hydroxy- oramino-substituted alkyl or benzyl. In another embodiment, each R^(L) ishydrogen, R^(y) is —OR^(c), —C(R^(a))₃ or —NHR^(c) where each R^(c) isindependently alkyl, substituted alkyl, aryl or substituted aryl, forinstance hydroxy- or amino-substituted alkyl or aryl; and R^(a) andR^(b) are independently hydrogen, alkyl, substituted alkyl, benzyl orsubstituted benzyl, for instance hydroxy- or amino-substituted alkyl orbenzyl. In a further embodiment, each R^(L) is hydrogen, R^(a) and R^(b)are independently benzyl or substituted alkyl. In a further embodiment,R^(y) is selected from the group consisting of alkyl and hydroxyalkyl.In certain embodiments, each R^(L) is hydrogen and R^(y) is—C(CH₃)₂CH₂OH.

In certain embodiments, 2-deoxy-2-fluoro-2-C-ethynyl-β-D-nucleosides canbe formed and derived into phosphoramidate compounds to potentiatedelivery of an active monophosphate to the liver of an individualinflicted with HCV, such as the compounds described herein by way ofexample. In certain embodiments, a compound of the following formula isprovided:

wherein:

T=O, S, CH₂, CH(hal) or CH(hal)₂, S(O)_(n);

n=1, 2;

hal=halogen;

R═H, acyl (with lower linear and non linear alkyl —C1 to 6-, aminoacid),monophosphate, diphosphate, triphosphate, monophosphate prodrug such as(alkyl-O)₂PO, (tBuSate-O)₂PO, cyclic monophosphate prodrug,phosphoramidate prodrug (aromatic amine, aminoacid);

X and Y are independently H, OH, O-alkyl (lower), O-acyl, F, NH₂,NH-alkyl, N-dialkyl, NH-acyl, N-diacyl, or azido;

Z is H, alkyl, alkenyl, alkynyl, hydroxymethyl, fluoromethyl, or azido;

W is H, alkyl, alkenyl, alkynyl, hydroxymethyl, fluoromethyl, azido,carboxylic acid, CO₂-alkyl, cyano, or carboxamide;

A is H, alkyl, alkenyl, alkynyl, hydroxymethyl, fluoromethyl, azido,carboxylic acid, CO₂-alkyl, cyano, or carboxamide; and

Base is a natural or modified base.

Optionally the compounds include a chlorine atom at the 2′-position.

In another embodiment, the compound provided herein is a compound offormula:

wherein R^(a), R^(b) and R^(y) are as defined in Formula IIa or IIb; Ais H, alkyl, alkenyl, alkynyl, hydroxymethyl, fluoromethyl, azido,carboxylic acid, CO₂-alkyl, cyano, or carboxamide; and R^(bl) is halo,alkoxy or haloalkyl. Each R^(L) is independently H, carbamyl, straightchained, branched or cyclic alkyl; acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester such as alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted; alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, a lipid,such as a phospho lipid; an amino acid; an amino acid residue; or acarbohydrate. In certain embodiments according to this paragraph, eachR^(L) is hydrogen, R^(y) is substituted alkyl, e.g. hydroxyalkyl oraminoalkyl; and R^(a) and R^(b) are independently hydrogen, alkyl,substituted alkyl, benzyl or substituted benzyl, for instance hydroxy-or amino-substituted alkyl or benzyl. In another embodiment, each R^(L)is hydrogen, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c) where each R^(c)is independently alkyl, substituted alkyl, aryl or substituted aryl, forinstance hydroxy- or amino-substituted alkyl or aryl; and R^(a) andR^(b) are independently hydrogen, alkyl, substituted alkyl, benzyl orsubstituted benzyl, for instance hydroxy- or amino-substituted alkyl orbenzyl. In a further embodiment, each R^(L) is hydrogen, R^(a) and R^(b)are independently benzyl or substituted alkyl. In a further embodiment,R^(y) is selected from the group consisting of alkyl and hydroxyalkyl.In certain embodiments, each R^(L) is hydrogen and R^(y) is—C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

wherein R^(a), R^(b) and R^(y) are as defined in Formula IIa or IIb. Incertain embodiments, R^(a) is hydrogen, R^(b) is —CH₂—C₆H₅ and R^(y) is—C(CH₃)₂CH₂OH. In certain embodiments according to this paragraph, R^(y)is substituted alkyl, e.g. hydroxyalkyl or aminoalkyl; and R^(a) andR^(b) are independently hydrogen, alkyl, substituted alkyl, benzyl orsubstituted benzyl, for instance hydroxy- or amino-substituted alkyl orbenzyl. In another embodiment, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c)where each R^(c) is independently alkyl, substituted alkyl, aryl orsubstituted aryl, for instance hydroxy- or amino-substituted alkyl oraryl; and R^(a) and R^(b) are independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl, for instance hydroxy- oramino-substituted alkyl or benzyl. In a further embodiment, R^(a) andR^(b) are independently benzyl or substituted alkyl. In a furtherembodiment, R^(y) is selected from the group consisting of alkyl andhydroxyalkyl. In certain embodiments, R^(y) is —C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

wherein X is halogen, R^(a), R^(b) and R^(y) are as defined in FormulaIIa or IIb and R² and R³ are each independently H, straight chained,branched or cyclic alkyl; acyl (including lower acyl); CO-alkyl,CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonateester such as alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted;alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, such as aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich is capable of providing a compound wherein R² and/or R³ isindependently H or phosphate (including mono-, di- or triphosphate), forexample when administered in vivo; or R² and R³ are linked to form acyclic group by an alkyl, ester or carbamate linkage. R^(L) is hydrogenor any lipophilic group known to those of skill in the art. In certainembodiments, R² and R³ are each hydrogen, R^(a) is hydrogen, R^(b) is—CH₂—C₆H₅ and R^(y) is —C(CH₃)₂CH₂OH. In certain embodiments saidlipophilic group is selected from alkyl, alkenyl, cycloalkyl, aryl,heteroaryl, arylalkyl and heteroaryl-alkyl. In certain embodimentsaccording to this paragraph, X is fluoro, R^(L) is hydrogen, R² and R³are each H, R^(y) is substituted alkyl, e.g. hydroxyalkyl or aminoalkyl;and R^(a) and R^(b) are independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl, for instance hydroxy- oramino-substituted alkyl or benzyl. In another embodiment, X is fluoro,R^(L) is hydrogen, R² and R³ are each H, R^(y) is —OR^(c), —C(R^(c))₃ or—NHR^(c) where each R^(c) is independently alkyl, substituted alkyl,aryl or substituted aryl, for instance hydroxy- or amino-substitutedalkyl or aryl; and R^(a) and R^(b) are independently hydrogen, alkyl,substituted alkyl, benzyl or substituted benzyl, for instance hydroxy-or amino-substituted alkyl or benzyl. In a further embodiment, X isfluoro, R^(L) is hydrogen, R² and R³ are each H, R^(a) and R^(b) areindependently benzyl or substituted alkyl. In a further embodiment, X isfluoro, R^(L) is hydrogen, R² and R³ are each H, R^(y) is selected fromthe group consisting of alkyl and hydroxyalkyl. In certain embodiments,X is fluoro, R^(L) is hydrogen, R² and R³ are each H, R^(y) is—C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

wherein R^(a), R^(b) and R^(y) are as defined in Formula IIa or IIb. Incertain embodiments, R^(y) is substituted alkyl, e.g. hydroxyalkyl oraminoalkyl; and R^(a) and R^(b) are independently hydrogen, alkyl,substituted alkyl, benzyl or substituted benzyl, for instance hydroxy-or amino-substituted alkyl or benzyl. In another embodiment, R^(y) is—OR^(c), —C(R^(c))₃ or —NHR^(c) where each R^(c) is independently alkyl,substituted alkyl, aryl or substituted aryl, for instance hydroxy- oramino-substituted alkyl or aryl; and R^(a) and R^(b) are independentlyhydrogen, alkyl, substituted alkyl, benzyl or substituted benzyl, forinstance hydroxy- or amino-substituted alkyl or benzyl. In a furtherembodiment, R^(a) and R^(b) are independently benzyl or substitutedalkyl. In a further embodiment, R^(y) is selected from the groupconsisting of alkyl and hydroxyalkyl. In certain embodiments, R^(a) ishydrogen, R^(b) is —C₁₋₁₂—C₆H₅ and R^(y) is —C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

wherein R^(a), R^(b) and R^(y) are as defined in Formula IIa or IIb. Incertain embodiments, R^(y) is substituted alkyl, e.g. hydroxyalkyl oraminoalkyl; and R^(a) and R^(b) are independently hydrogen, alkyl,substituted alkyl, benzyl or substituted benzyl, for instance hydroxy-or amino-substituted alkyl or benzyl. In another embodiment, R^(y) is—OR^(c), —C(R^(c))₃ or —NHR^(c) where each R^(c) is independently alkyl,substituted alkyl, aryl or substituted aryl, for instance hydroxy- oramino-substituted alkyl or aryl; and R^(a) and R^(b) are independentlyhydrogen, alkyl, substituted alkyl, benzyl or substituted benzyl, forinstance hydroxy- or amino-substituted alkyl or benzyl. In a furtherembodiment, R^(a) and R^(b) are independently benzyl or substitutedalkyl. In a further embodiment, R^(y) is selected from the groupconsisting of alkyl and hydroxyalkyl. In certain embodiments, R^(a) ishydrogen, R^(b) is —CH₂—C₆H₅ and R^(y) is —C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

wherein R^(a), R^(b) and R^(y) are as defined in Formula IIa or IIb. Incertain embodiments, R^(y) is substituted alkyl, e.g. hydroxyalkyl oraminoalkyl; and R^(a) and R^(b) are independently hydrogen, alkyl,substituted alkyl, benzyl or substituted benzyl, for instance hydroxy-or amino-substituted alkyl or benzyl. In another embodiment, R^(y) is—OR^(c), —C(R^(c))₃ or —NHR^(c) where each R^(c) is independently alkyl,substituted alkyl, aryl or substituted aryl, for instance hydroxy- oramino-substituted alkyl or aryl; and R^(a) and R^(b) are independentlyhydrogen, alkyl, substituted alkyl, benzyl or substituted benzyl, forinstance hydroxy- or amino-substituted alkyl or benzyl. In a furtherembodiment, R^(a) and R^(b) are independently benzyl or substitutedalkyl. In a further embodiment, R^(y) is selected from the groupconsisting of alkyl and hydroxyalkyl. In certain embodiments, R^(a) ishydrogen, R^(b) is —CH₂—C₆H₅ and R^(y) is —C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

wherein R^(a), R^(b) and R^(y) are as defined in Formula IIa or IIb. Incertain embodiments, R^(y) is substituted alkyl, e.g. hydroxyalkyl oraminoalkyl; and R^(a) and R^(b) are independently hydrogen, alkyl,substituted alkyl, benzyl or substituted benzyl, for instance hydroxy-or amino-substituted alkyl or benzyl. In another embodiment, R^(y) is—OR^(c), —C(R^(c))₃ or —NHR^(c) where each R^(c) is independently alkyl,substituted alkyl, aryl or substituted aryl, for instance hydroxy- oramino-substituted alkyl or aryl; and R^(a) and R^(b) are independentlyhydrogen, alkyl, substituted alkyl, benzyl or substituted benzyl, forinstance hydroxy- or amino-substituted alkyl or benzyl. In a furtherembodiment, R^(a) and R^(b) are independently benzyl or substitutedalkyl. In a further embodiment, R^(y) is selected from the groupconsisting of alkyl and hydroxyalkyl. In certain embodiments, R^(a) ishydrogen, R^(b) is —CH₂—C₆H₅ and R^(y) is —C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

wherein R^(a), R^(b) and R^(y) are as defined in Formula IIa or IIb.R^(L) is hydrogen or any lipophilic group known to those of skill in theart. In certain embodiments, R^(a) is hydrogen, R^(b) is —CH₂—C₆H₅ andR^(y) is —C(CH₃)₂CH₂OH. In certain embodiments said lipophilic group isselected from alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, arylalkyland heteroaryl-alkyl. In certain embodiments according to thisparagraph, R^(y) is substituted alkyl, e.g. hydroxyalkyl or aminoalkyl;and R^(a) and R^(b) are independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl, for instance hydroxy- oramino-substituted alkyl or benzyl. In another embodiment, R^(y) is—C(R^(c))₃ or —NHR^(c) where each R^(c) is independently alkyl,substituted alkyl, aryl or substituted aryl, for instance hydroxy- oramino-substituted alkyl or aryl; and R^(a) and R^(b) are independentlyhydrogen, alkyl, substituted alkyl, benzyl or substituted benzyl, forinstance hydroxy- or amino-substituted alkyl or benzyl. In a furtherembodiment, R^(a) and R^(b) are independently benzyl or substitutedalkyl. In a further embodiment, R^(y) is selected from the groupconsisting of alkyl and hydroxyalkyl. In certain embodiments, R^(y) is—C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

wherein the variables are as described above.

In another embodiment, the compound provided herein is a compound offormula:

wherein the variables are as described above.

In one embodiment, R¹ is a natural nucleoside. In one embodiment, R¹ isa 2′- or 3′-prodrug of biologically active 1′, 2′, 3′ or 4′C-branchedβ-D or β-L nucleoside. The term 1′, 2′, 3′ or 4′C-branched, as used inthis specification, includes a nucleoside that has an additionalnon-natural substituent in the 1′, 2′, 3′ or 4′-position (i.e., carbonis bound to four nonhydrogen substituents). The term 2′-prodrug, as usedherein, includes a 1′, 2′, 3′ or 4′ C-branched-β-D or β-L nucleosidethat has a biologically cleavable moiety at the 2′-position, including,but not limited to acyl, and in one embodiment, a natural or synthetic Dor L amino acid, in one embodiment, an L-amino acid. The term3′-prodrug, as used herein, includes a 1′, 2′, 3′ or 4′ C-branched-β-Dor β-L nucleoside that has a biologically cleavable moiety at the3′-position, including, but not limited to acyl, and in one embodiment,a natural or synthetic D or L amino acid, in one embodiment, an L-aminoacid. In one embodiment, the amino acid is valine.

Examples of prodrugs (that can be further derivatized as describedherein to include a phosphoramidate or phosphonoamidate moiety, forexample, at the 5′ position) include 2′-L-valine ester ofβ-D-2′-C-methyl-cytidine; 2′-L-valine ester ofβ-D-2′-C-methyl-thymidine; 2′-L-valine ester ofβ-D-2′-C-methyl-adenosine; 2′-L-valine ester ofβ-D-2′-C-methyl-guanosine; 2′-L-valine ester ofβ-D-2′-C-methyl-5-fluorocytidine; 2′-L-valine ester ofβ-D-2′-C-methyl-uridine; 2′-acetyl ester of β-D-2′-C-methyl-cytidine;2′-acetyl ester of β-D-2′-C-methyl-thymidine; 2′-acetyl ester ofβ-D-2′-C-methyl-adenosine; 2′-acetyl ester of β-D-2′-C-methyl-guanosine;2′-acetyl ester of β-D-2′-C-methyl-5-fluoro-cytidine; and 2′-esters ofβ-D-2′-C-methyl-(cytidine, 5-fluorocytidine, guanosine, uridine,adenosine, or thymidine) wherein (i) the 2′ ester is an amino acidester; or (ii) the 2′ ester is an alkyl or aryl ester.

Further examples of prodrugs are 3′-L-valine ester ofβ-D-2′-C-methyl-cytidine; 3′-L-valine ester ofβ-D-2′-C-methyl-thymidine; 3′-L-valine ester ofβ-D-2′-C-methyl-adenosine; 3′-L-valine ester ofβ-D-2′-C-methyl-guanosine; 3′-L-valine ester ofβ-D-2′-C-methyl-5-fluorocytidine; 3′-L-valine ester ofβ-D-2′-C-methyl-uridine; 3′-acetyl ester of β-D-2′-C-methyl-cytidine;3′-acetyl ester of β-D-2′-C-methyl-thymidine; 3′-acetyl ester ofβ-D-2′-C-methyl-adenosine; 3′-acetyl ester of β-D-2′-C-methyl-guanosine;3′-acetyl ester of 13-D-2′-C-methyl-5-fluoro-cytidine; and 3′-esters ofβ-D-2′-C-methyl-(cytidine, 5-fluorocytidine, guanosine, uridine,adenosine, or thymidine) wherein (i) the 3′ ester is an amino acidester; or (ii) the 3′ ester is an alkyl or aryl ester.

Additional examples of prodrugs include 2′,3′-L-divaline ester ofβ-D-2′-C-methyl-cytidine (dival-2′-Me-L-dC); 2′,3′-L-divaline ester ofβ-D-2′-C-methyl-thymidine; 2′,3′-L-divaline ester ofβ-D-2′-C-methyl-adenosine; 2′,3′-L-divaline ester ofβ-D-2′-C-methyl-guanosine; 2′,3′-L-divaline ester ofβ-D-2′-C-methyl-5-fluoro-cytidine; 2′,3′-L-divaline ester ofβ-D-2′-C-methyl-uridine; 2′,3′-diacetyl ester ofβ-D-2′-C-methyl-cytidine; 2′,3′-diacetyl ester ofβ-D-2′-C-methyl-thymidine; 2′,3′-diacetyl ester ofβ-D-2′-C-methyl-adenosine; 2′,3′-diacetyl ester ofβ-D-2′-C-methyl-guanosine; 2′,3′-diacetyl ester ofβ-D-2′-C-methyl-5-fluoro-cytidine; and 2′,3′-diesters ofβ-D-2′-C-methyl-(cytidine, 5-fluorocytidine, guanosine, uridine,adenosine, or thymidine) wherein (i) the 2′ ester is an amino acid esterand the 3′-ester is an alkyl or aryl ester; (ii) both esters are aminoacid esters; (iii) both esters are independently alkyl or aryl esters;or (iv) the 2′ ester is an alkyl or aryl ester and the 3′-ester is anamino acid ester.

In one embodiment, R¹ is:

wherein Base is a natural or non-natural purine or pyrimidine base asdefined herein;

R⁶ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, azido, cyano,Br-vinyl, alkoxy, acyloxy, alkoxycarbonyl, alkenyloxy, halo, NO₂ orNR^(6a)R^(6b);

R^(6a) and R^(6b) are each independently hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, acyl, aryl, heteroaryl or heterocyclyl;

R⁷, R⁹, R⁸ and R¹⁰ are selected as follows:

i) R⁷ and R⁹ are each independently hydrogen, OR^(7a), hydroxy, alkyl,alkenyl, alkynyl, azido, cyano, Br-vinyl, alkyloxycarbonyl, acyloxy,halo, NO₂ or NR^(6a)R⁶b;

ii) R⁸ and R¹⁰ are each independently H, alkyl or halo; or

iii) each R⁷ and R⁹, R⁷ and R¹⁰, R⁸ and R⁹ or R⁸ and R¹⁰ together form adouble bond;

R^(7a) is H; straight chained, branched or cyclic alkyl (including loweralkyl); acyl (including lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl,CO-aryloxyalkyl, CO-substituted aryl, sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; alkylsulfonyl,arylsulfonyl, arylalkylsulfonyl, a lipid, including a phospholipid; anamino acid; and amino acid residue, a carbohydrate; a peptide;cholesterol; or other pharmaceutically acceptable leaving group which iscapable of providing a compound wherein R^(7a) is H or phosphate(including mono-, di- or triphosphate), for example, when administeredin vivo; wherein in one embodiment R^(7a) is not phosphate (includingmono-, di- or triphosphate or a stabilized phosphate prodrug), or twoR^(7a) groups are linked to form a cyclic group by an alkyl, ester orcarbamate linkage; and

X is O, S, SO₂ or CH₂.

In one embodiment, R¹ has formula:

wherein and R² and R³ are each independently H; straight chained,branched or cyclic alkyl; acyl (including lower acyl); CO-alkyl,CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonateester such as alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted;alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, a lipid, such as aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich is capable of providing a compound wherein R² and/or R³ isindependently H or phosphate (including mono-, di- or triphosphate), forexample when administered in vivo; or R² and Ware linked to form acyclic group by an alkyl, ester or carbamate linkage;

wherein Y¹ is hydrogen, bromo, chloro, fluoro, iodo, CN, OH, OR⁴, NH₂,NHR⁴, NR⁴R⁵, SH or SR⁴;

X¹ is a straight chained, branched or cyclic optionally substitutedalkyl, CH₃, CF₃, C(Y³)₃, 2-Br-ethyl, CH₂F, CH₂Cl, CH₂CF₃, CF₂CF₃,C(Y³)₂C(Y³)₃, CH₂OH, optionally substituted alkenyl, optionallysubstituted alkynyl, COOH, COOR⁴, COO-alkyl, COO-aryl, CO-Oalkoxyalkyl,CONH₂, CONHR⁴, CON(R⁴)₂, chloro, bromo, fluoro, iodo, CN, N₃, OH, OR⁴,NH₂, NHR⁴, NR⁴R⁵, SH or SR⁵; and

X² is H, straight chained, branched or cyclic optionally substitutedalkyl, CH₃, CF₃, C(Y³)₃, 2-Br-ethyl, CH₂F, CH₂Cl, CH₂CF₃, CF₂CF₃,C(Y³)₂C(Y³)₃, CH₂OH, optionally substituted alkenyl, optionallysubstituted alkynyl, COOH, COOR⁴, COO-alkyl, COO-aryl, CO-Oalkoxyalkyl,CONH₂, CONHR⁴, CON(R⁴)₂, chloro, bromo, fluoro, iodo, CN, N₃, OH, OR⁴,NH₂, NHR⁴, NR⁴R⁵, SH or SR⁵; and

wherein each Y³ is independently H, F, Cl, Br or I;

each R⁴ and R⁵ is independently hydrogen, acyl (including lower acyl),alkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl), lower alkyl, alkenyl, alkynyl or cycloalkyl.

In the embodiments described herein, R² and/or R³ may be apharmaceutically acceptable leaving group which is capable of providinga compound wherein R² and/or R³ is independently H or phosphate(including mono-, di- or triphosphate), for example when administered invivo.

In another embodiment, each R² and R³ is independently hydrogen or acyl.In another embodiment, R² and R³ are linked to form a cyclic group by analkyl, ester or carbamate linkage.

In another embodiment, R¹ is:

wherein R², R³, Y¹, Y³, X¹ and X² are as defined in Formula XIII.

In one embodiment, R¹ is:

wherein Base is selected from the group consisting of

wherein each W¹, W², W³ and W⁴ is independently N, CH, CF, Cl, CBr, CCl,CCN, CCH₃, CCF₃, CCH₂CH₃, CC(O)NH₂, CC(O)NHR⁴, CC(O)N(R⁴)₂, CC(O)OH,CC(O)OR⁴ or CX³;

each W* is independently O, S, NH or NR⁴;

X is O, S, SO₂, CH₂, CH₂OH, CHF, CF₂, C(Y³)₂, CHCN, C(CN)₂, CHR⁴ orC(R⁴)₂;

X* is CH, CF, CY³ or CR⁴;

X² is H, straight chained, branched or cyclic optionally substitutedalkyl, CH₃, CF₃, C(Y³)₃, 2-Br-ethyl, CH₂F, CH₂Cl, CH₂CF₃, CF₂CF₃,C(Y³)₂C(Y³)₃, CH₂OH, optionally substituted alkenyl, optionallysubstituted alkynyl, COOH, COOR⁴, COO-alkyl, COO-aryl, CO-Oalkoxyalkyl,CONH₂, CONHR⁴, CON(R⁴)₂, chloro, bromo, fluoro, iodo, CN, N₃, OH, OR⁴,NH₂, NHR⁴, NR⁴R⁵, SH or SR⁵;

each X³ is independently a straight chained, branched or cyclicoptionally substituted alkyl (including lower alkyl), CH₃, CH₂CN, CH₂N₃,CH₂NH₂, CH₂NHCH₃, CH₂N(CH₃)₂, CH₂OH, halogenated alkyl (includinghalogenated lower alkyl), CF₃, C(Y³)₃, 2-Br-ethyl, CH₂F, CH₂Cl, CH₂CF₃,CF₂CF₃, C(Y³)₂C(Y³)₃, optionally substituted alkenyl, haloalkenyl,Br-vinyl, optionally substituted alkynyl, haloalkynyl, N₃, CN, —C(O)OH,—C(O)OR⁴, —C(O)O(lower alkyl), —C(O)NH₂, —C(O)NHR⁴, —C(O)NH(lower—C(O)N(R⁴)₂, —C(O)N(lower alkyl)₂, OH, OR⁴, —O(acyl), —O(lower acyl),—O(alkyl), —O(lower alkyl), —O(alkenyl), —O(alkynyl), —O(arylalkyl),—O(cycloalkyl), —S(acyl), —S(lower acyl), —S(R⁴), —S(lower alkyl),—S(alkenyl), —S(alkynyl), —S(arylalkyl), —S(cycloalkyl), chloro, bromo,fluoro, iodo, NH₂, —NH(lower alkyl), —NHR⁴, —NR⁴R⁵, —NH(acyl), —N(loweralkyl)₂, —NH(alkenyl), —NH(alkynyl), —NH(arylalkyl), —NH(cycloalkyl),—N(acyl)₂;

each Y is independently selected from the group consisting of H,optionally substituted lower alkyl, cycloalkyl, alkenyl, alkynyl, CH₂OH,CH₂NH₂, CH₂NHCH₃, CH₂N(CH₃)₂, CH₂F, CH₂Cl, CH₂N₃, CH₂CN, CH₂CF₃, CF₃,CF₂CF₃, CH₂CO₂R, (CH₂)_(m)CO—OH, (CH₂)_(m)COOR, (CH₂)_(m)CONH₂,(CH₂)_(m)CONR₂, and (CH₂)_(m)CONHR;

wherein R is H, alkyl or acyl;

Y¹ is hydrogen, bromo, chloro, fluoro, iodo, CN, OH, OR⁴, NH₂, NHR⁴,NR⁴R⁵, SH or SR⁴;

each Y² is independently O, S, NH or NR⁴;

each Y³ is independently H, F, Cl, Br or I;

each R⁴ and R⁵ is independently hydrogen, acyl (including lower acyl),alkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl), lower alkyl, alkenyl, alkynyl or cycloalkyl;

each R⁶ is independently an optionally substituted alkyl (includinglower alkyl), CH₃, CH₂CN, CH₂N₃, CH₂NH₂, CH₂NHCH₃, CH₂N(CH₃)₂, CH₂OH,halogenated alkyl (including halogenated lower alkyl), CF₃, C(Y³)₃,2-Br-ethyl, CH₂F, CH₂Cl, CH₂CF₃, CF₂CF₃, C(Y³)₂C(Y³)₃, optionallysubstituted alkenyl, haloalkenyl, Br-vinyl, optionally substitutedalkynyl, haloalkynyl, —CH₂C(O)OH, —CH₂C(O)OR⁴, —CH₂C(O)O(lower alkyl),—CH₂C(O)NH₂, —CH₂C(O)NHR⁴, —CH₂C(O)NH(lower alkyl), —CH₂C(O)N(R⁴)₂,—CH₂C(O)N(lower alkyl)₂, —(CH₂)_(m)C(O)OH, —(CH₂)_(m)C(O)OR⁴,—(CH₂)_(m)C(O)O(lower alkyl), —(CH₂)_(m)C(O)NH₂, —(CH₂)_(m)C(O)NHR⁴,—(CH₂)_(m)C(O)NH(lower alkyl), —(CH₂)_(m)C(O)N(R⁴)₂,—(CH₂)_(m)C(O)N(lower alkyl)₂, —C(O)OH, —C(O)OR⁴, —C(O)O(lower alkyl),—C(O)NH₂, —C(O)NHR⁴, —C(O)NH(lower alkyl), —C(O)N(R⁴)₂, —C(O)N(loweralkyl)₂ or cyano;

each R⁷ is independently H, OH, OR², optionally substituted alkyl(including lower alkyl), CH₃, CH₂CN, CH₂N₃, CH₂NH₂, CH₂NHCH₃,CH₂N(CH₃)₂, CH₂OH, halogenated alkyl (including halogenated loweralkyl), CF₃, C(Y³)₃, 2-Br-ethyl, CH₂F, CH₂Cl, CH₂CF₃, CF₂CF₃,C(Y³)₂C(Y³)₃, optionally substituted alkenyl, haloalkenyl, Br-vinyl,optionally substituted alkynyl, haloalkynyl, optionally substitutedcarbocycle (for example, a 3-7 membered carbocyclic ring), optionallysubstituted heterocycle (for example, a 3-7 membered heterocyclic ringhaving one or more O, S and/or N), optionally substituted heteroaryl(for example, a 3-7 membered heteroaromatic ring having one or more O, Sand/or N), —CH₂C(O)OH, —CH₂C(O)OR⁴, —CH₂C(O)O(lower alkyl), —CH₂C(O)SH,—CH₂C(O)SR⁴, —CH₂C(O)S(lower alkyl), —CH₂C(O)NH₂, —CH₂C(O)NHR⁴,—CH₂C(O)NH(lower alkyl), —CH₂C(O)N(R⁴)₂, —CH₂C(O)N(lower alkyl)₂,—(CH₂)_(m)C(O)OH, —(CH₂)_(m)C(O)OR⁴, —(CH₂)_(m)C(O)O(lower alkyl),—(CH₂)_(m)C(O)SH, —(CH₂)_(m)C(O)SR⁴, —(CH₂)_(m)C(O)S(lower alkyl),—(CH₂)_(m)C(O)NH₂, —(CH₂)_(m)C(O)NHR⁴, —(CH₂)_(m)C(O)NH(lower alkyl),—(CH₂)_(m)C(O)N(R⁴)₂, —(CH₂)_(m)C(O)N(lower alkyl)₂, —C(O)OH, —C(O)OR⁴,—C(O)O(lower alkyl), —C(O)SH, —C(O)SR⁴, —C(O)S(lower alkyl), —C(O)NH₂,—C(O)NHR⁴, —C(O)NH(lower alkyl), —C(O)N(R⁴)₂, —C(O)N(lower alkyl)₂,—O(acyl), —O(lower acyl), —O(R⁴), —O(alkyl), —O(lower alkyl),—O(alkenyl), —O(alkynyl), —O(arylalkyl), —O(cycloalkyl), —S(acyl),—S(lower acyl), —S(R⁴), —S(lower alkyl), —S(alkenyl), —S(alkynyl),—S(arylalkyl), —S(cycloalkyl), NO₂, NH₂, —NH(lower alkyl), —NHR⁴,—NR⁴R⁵, —NH(acyl), —N(lower alkyl)₂, —NH(alkenyl), —NH(alkynyl),—NH(arylalkyl), —NH(cycloalkyl), —N(acyl)₂, azido, cyano, SCN, OCN, NCOor halo (fluoro, chloro, bromo, iodo);

alternatively, R⁶ and R⁷ can come together to form a spiro compoundselected from the group consisting of optionally substituted carbocycle(for example, a 3-7 membered carbocyclic ring) or optionally substitutedheterocycle (for example, a 3-7 membered heterocyclic ring having one ormore O, S and/or N);

each m is independently 0, 1 or 2.

In one embodiment, the base is

In one embodiment, the base is

In another embodiment, R¹ is

wherein each R⁶ and R⁷ is as defined in Formulae XX, XXI or XXII above;

wherein each R⁸ and R¹¹ is independently hydrogen, an optionallysubstituted alkyl (including lower alkyl), CH₃, CH₂CN, CH₂N₃, CH₂NH₂,CH₂NHCH₃, CH₂N(CH₃)₂, CH₂OH, halogenated alkyl (including halogenatedlower alkyl), CF₃, C(Y³)₃, 2-Br-ethyl, CH₂F, CH₂Cl, CH₂CF₃, CF₂CF₃,C(Y³)₂C(Y³)₃, optionally substituted alkenyl, haloalkenyl, Br-vinyl,optionally substituted alkynyl, haloalkynyl, —CH₂C(O)OH, —CH₂C(O)OR⁴,—CH₂C(O)O(lower alkyl), —CH₂C(O)NH₂, —CH₂C(O)NHR⁴, —CH₂C(O)NH(loweralkyl), —CH₂C(O)N(R⁴)₂, —CH₂C(O)N(lower alkyl)₂, —(CH₂)_(m)C(O)OH,—(CH₂)_(m)C(O)OR⁴, —(CH₂)_(m)C(O)O(lower alkyl), —(CH₂)_(m)C(O)NH₂,—(CH₂)_(m)C(O)NHR⁴, —(CH₂)_(m)C(O)NH(lower alkyl), —(CH₂)_(m)C(O)NR⁴)₂,—(CH₂)_(m)C(O)N(lower alkyl)₂, —C(O)OH, —C(O)OR⁴, —C(O)O(lower alkyl),—C(O)NH₂, —C(O)NHR⁴, —C(O)NH(lower alkyl), —C(O)N(R⁴)₂, —C(O)N(loweralkyl)₂, cyano, azido, NH-acyl or N(acyl)₂;

each R⁹ and R¹⁰ are independently hydrogen, OH, OR², optionallysubstituted alkyl (including lower alkyl), CH₃, CH₂CN, CH₂N₃, CH₂NH₂,CH₂NHCH₃, CH₂N(CH₃)₂, CH₂OH, halogenated alkyl (including halogenatedlower alkyl), CF₃, C(Y³)₃, 2-Br-ethyl, CH₂F, CH₂Cl, CH₂CF₃, CF₂CF₃,C(Y³)₂C(Y³)₃, optionally substituted alkenyl, haloalkenyl, Br-vinyl,optionally substituted alkynyl, haloalkynyl, optionally substitutedcarbocycle (for example, a 3-7 membered carbocyclic ring), optionallysubstituted heterocycle (for example, a 3-7 membered heterocyclic ringhaving one or more O, S and/or N), optionally substituted heteroaryl(for example, a 3-7 membered heteroaromatic ring having one or more O, Sand/or N), —CH₂C(O)OH, —CH₂C(O)OR⁴, —CH₂C(O)O(lower alkyl), —CH₂C(O)SH,—CH₂C(O)SR⁴, —CH₂C(O)S(lower alkyl), —CH₂C(O)NH₂, —CH₂C(O)NHR⁴,—CH₂C(O)NH(lower alkyl), —CH₂C(O)N(R⁴)₂, —CH₂C(O)N(lower alkyl)₂,—(CH₂)_(m)C(O)OH, —(CH₂)_(m)C(O)OR⁴, —(CH₂)_(m)C(O)O(lower alkyl),—(CH₂)_(m)C(O)SH, —(CH₂)_(m)C(O)SR⁴, —(CH₂)_(m)C(O)S(lower alkyl),—(CH₂)_(m)C(O)NH₂, —(CH₂)_(m)C(O)NHR⁴, —(CH₂)_(m)C(O)NH(lower alkyl),—(CH₂)_(m)C(O)N(R⁴)₂, —(CH₂)_(m)C(O)N(lower alkyl)₂, —C(O)OH, —C(O)OR⁴,—C(O)O(lower alkyl), —C(O)SH, —C(O)SR⁴, —C(O)S(lower alkyl), —C(O)NH₂,—C(O)NHR⁴, —C(O)NH(lower alkyl), —C(O)N(R⁴)₂, —C(O)N(lower alkyl)₂,—O(acyl), —O(lower acyl), —O(R⁴), —O(alkyl), —O(lower alkyl),—O(alkenyl), —O(alkynyl), —O(arylalkyl), —O(cycloalkyl), —S(acyl),—S(lower acyl), —S(R⁴), —S(lower alkyl), —S(alkenyl), —S(alkynyl),—S(arylalkyl), —S(cycloalkyl), NO₂, NH₂, —NH(lower alkyl), —NHR⁴,—NR⁴R⁵, —NH(acyl), —N(lower alkyl)₂, —NH(alkenyl), —NH(alkynyl),—NH(arylalkyl), —NH(cycloalkyl), —N(acyl)₂, azido, cyano, SCN, OCN, NCOor halo (fluoro, chloro, bromo, iodo);

each m is independently 0, 1 or 2;

alternatively, R⁶ and R¹⁰, R⁷ and R⁹, R⁸ and R⁷ or R⁹ and R¹¹ can cometogether to form a bridged compound selected from the group consistingof optionally substituted carbocycle (for example, a 3-7 memberedcarbocyclic ring) or optionally substituted heterocycle (for example, a3-7 membered heterocyclic ring having one or more O, S and/or N); or

alternatively, R⁶ and R⁷ or R⁹ and R¹⁰ can come together to form a spirocompound selected from the group consisting of optionally substitutedcarbocycle (for example, a 3-7 membered carbocyclic ring) or optionallysubstituted heterocycle (for example, a 3-7 membered heterocyclic ringhaving one or more O, S and/or N).

In another embodiment, R¹ is:

wherein Base* is a purine or pyrimidine base as defined herein;

each R¹² is independently a substituted alkyl (including lower alkyl),CH₂CN, CH₂N₃, CH₂NH₂, CH₂NHCH₃, CH₂N(CH₃)₂, CH₂OH, halogenated alkyl(including halogenated lower alkyl), CF₃, C(Y³)₃, 2-Br-ethyl, CH₂F,CH₂Cl, CH₂CF₃, CF₂CF₃, C(Y³)₂C(Y³)₃, substituted alkenyl, haloalkenyl(but not Br-vinyl), substituted alkynyl, haloalkynyl, —CH₂C(O)OH,—CH₂C(O)OR⁴, —CH₂C(O)O(lower alkyl), —CH₂C(O)NH₂, —CH₂C(O)NHR⁴,—CH₂C(O)NH(lower alkyl), —CH₂C(O)N(R⁴)₂, —CH₂C(O)N(lower alkyl)₂,—(CH₂)_(m)C(O)OH, —(CH₂)_(m)C(O)OR⁴, —(CH₂)_(m)C(O)O(lower alkyl),—(CH₂)_(m)C(O)NH₂, —(CH₂)_(m)C(O)NHR⁴, —(CH₂)_(m)C(O)NH(lower alkyl),—(CH₂)_(m)C(O)N(R⁴)₂, —(CH₂)_(m)C(O)N(lower alkyl)₂, —C(O)OH, —C(O)OR⁴,—C(O)NH₂, —C(O)NHR⁴, —C(O)NH(lower alkyl), —C(O)N(R⁴)₂, —C(O)N(loweralkyl)₂;

each R¹³ is independently substituted alkyl (including lower alkyl),CH₂CN, CH₂N₃, CH₂NH₂, CH₂NHCH₃, CH₂N(CH₃)₂, CH₂OH, halogenated alkyl(including halogenated lower alkyl), CF₃, C(Y³)₃, 2-Br-ethyl, CH₂F,CH₂Cl, CH₂CF₃, CF₂CF₃, C(Y³)₂C(Y³)₃, substituted alkenyl, haloalkenyl(but not Br-vinyl), substituted alkynyl, haloalkynyl, optionallysubstituted carbocycle (for example, a 3-7 membered carbocyclic ring),optionally substituted heterocycle (for example, a 3-7 memberedheterocyclic ring having one or more O, S and/or N), optionallysubstituted heteroaryl (for example, a 3-7 membered heteroaromatic ringhaving one or more O, S and/or N), —CH₂C(O)OH, —CH₂C(O)OR⁴,—CH₂C(O)O(lower alkyl), —CH₂C(O)SH, —CH₂C(O)SR⁴, —CH₂C(O)S(lower alkyl),—CH₂C(O)NH₂, —CH₂C(O)NHR⁴, —CH₂C(O)NH(lower alkyl), —CH₂C(O)N(R⁴)₂,—CH₂C(O)N(lower alkyl)₂, —(CH₂)_(m)C(O)OH, —(CH₂)_(m)C(O)OR⁴,—(CH₂)_(m)C(O)O(lower alkyl), —(CH₂)_(m)C(O)SH, —(CH₂)_(m)C(O)SR⁴,—(CH₂)_(m)C(O)S(lower alkyl), —(CH₂)_(m)C(O)NH₂, —(CH₂)_(m)C(O)NHR⁴,—(CH₂)_(m)C(O)NH(lower alkyl), —(CH₂)_(m)C(O)N(R⁴)₂,—(CH₂)_(m)C(O)N(lower alkyl)₂, —C(O)OH, —C(O)OR⁴, —C(O)SH, —C(O)SR⁴,—C(O)S(lower alkyl), —C(O)NH₂, —C(O)NHR⁴, —C(O)NH(lower alkyl),—C(O)N(R⁴)₂, —C(O)N(lower alkyl)₂, —O(R⁴), —O(alkynyl), —O(arylalkyl),—O(cycloalkyl), —S(acyl), —S(lower acyl), —S(R⁴), —S(lower alkyl),—S(alkenyl), —S(alkynyl), —S(arylalkyl), —S(cycloalkyl), —NHR⁴, —NR⁴R⁵,—NH(alkenyl), —NH(alkynyl), —NH(arylalkyl), —NH(cycloalkyl), SCN, OCN,NCO or fluoro;

alternatively, R¹² and R¹³ can come together to form a spiro compoundselected from the group consisting of optionally substituted carbocycle(for example, a 3-7 membered carbocyclic ring) or optionally substitutedheterocycle (for example, a 3-7 membered heterocyclic ring having one ormore O, S and/or N);

R² and R³ are according to Formula XII; and

each m is independently 0, 1 or 2.

In another embodiment, R is:

wherein Base* is a purine or pyrimidine base as described herein; and

each R⁸ and R¹¹ is independently hydrogen, an optionally substitutedalkyl (including lower alkyl), CH₃, CH₂CN, CH₂N₃, CH₂NH₂, CH₂NHCH₃,CH₂N(CH₃)₂, CH₂OH, halogenated alkyl (including halogenated loweralkyl), CF₃, C(Y³)₃, 2-Br-ethyl, CH₂F, CH₂Cl, CH₂CF₃, CF₂CF₃,C(Y³)₂C(Y³)₃, optionally substituted alkenyl, haloalkenyl, Br-vinyl,optionally substituted alkynyl, haloalkynyl, —CH₂C(O)OH, —CH₂C(O)OR⁴,—CH₂C(O)O(lower alkyl), —CH₂C(O)NH₂, —CH₂C(O)NHR⁴, —CH₂C(O)NH(loweralkyl), —CH₂C(O)N(R⁴)₂, —CH₂C(O)N(lower alkyl)₂, —(CH₂)_(m)C(O)OH,—(CH₂)_(m)C(O)OR⁴, —(CH₂)_(m)C(O)O(lower alkyl), —(CH₂)_(m)C(O)NH₂,—(CH₂)_(m)C(O)NHR⁴, —(CH₂)_(m)C(O)NH(lower alkyl), —(CH₂)_(m)C(O)N(R⁴)₂,—(CH₂)_(m)C(O)N(lower alkyl)₂, —C(O)OH, —C(O)OR⁴, —C(O)O(lower alkyl),—C(O)NH₂, —C(O)NHR⁴, —C(O)NH(lower alkyl), —C(O)N(R⁴)₂, —C(O)N(loweralkyl)₂, cyano, NH-acyl or N(acyl)₂;

each R⁹ and R¹⁰ are independently hydrogen, OH, OR², optionallysubstituted alkyl (including lower alkyl), CH₃, CH₂CN, CH₂N₃, CH₂NH₂,CH₂NHCH₃, CH₂N(CH₃)₂, CH₂OH, halogenated alkyl (including halogenatedlower alkyl), CF₃, C(Y³)₃, 2-Br-ethyl, CH₂F, CH₂Cl, CH₂CF₃, CF₂CF₃,C(Y³)₂C(Y³)₃, optionally substituted alkenyl, haloalkenyl, Br-vinyl,optionally substituted alkynyl, haloalkynyl, optionally substitutedcarbocycle (for example, a 3-7 membered carbocyclic ring), optionallysubstituted heterocycle (for example, a 3-7 membered heterocyclic ringhaving one or more O, S and/or N), optionally substituted heteroaryl(for example, a 3-7 membered heteroaromatic ring having one or more O, Sand/or N), —CH₂C(O)OH, —CH₂C(O)OR⁴, —CH₂C(O)O(lower alkyl), —CH₂C(O)SH,—CH₂C(O)SR⁴, —CH₂C(O)S(lower alkyl), —CH₂C(O)NH₂, —CH₂C(O)NHR⁴,—CH₂C(O)NH(lower alkyl), —CH₂C(O)N(R⁴)₂, —CH₂C(O)N(lower alkyl)₂,—(CH₂)_(m)C(O)OH, —(CH₂)_(m)C(O)OR⁴, —(CH₂)_(m)C(O)O(lower alkyl),—(CH₂)_(m)C(O)SH, —(CH₂)_(m)C(O)SR⁴, —(CH₂)_(m)C(O)S(lower alkyl),—(CH₂)_(m)C(O)NH₂, —(CH₂)_(m)C(O)NHR⁴, —(CH₂)_(m)C(O)NH(lower alkyl),—(CH₂)_(m)C(O)N(R⁴)₂, —(CH₂)_(m)C(O)N(lower alkyl)₂, —C(O)OH, —C(O)OR⁴,—C(O)O(lower alkyl), —C(O)SH, —C(O)SR⁴, —C(O)S(lower alkyl), —C(O)NH₂,—C(O)NHR⁴, —C(O)NH(lower alkyl), —C(O)N(R⁴)₂, —C(O)N(lower alkyl)₂,—O(acyl), —O(lower acyl), —O(R⁴), —O(alkyl), —O(lower alkyl),—O(alkenyl), —O(alkynyl), —O(arylalkyl), —O(cycloalkyl), —S(acyl),—S(lower acyl), —S(R⁴), —S(lower alkyl), —S(alkenyl), —S(alkynyl),—S(arylalkyl), —S(cycloalkyl), NO₂, NH₂, —NH(lower alkyl), —NHR⁴,—NR⁴R⁵, —NH(acyl), —N(lower alkyl)₂, —NH(alkenyl), —NH(alkynyl),—NH(arylalkyl), —NH(cycloalkyl), —N(acyl)₂, azido, cyano, SCN, OCN, NCOor halo (fluoro, chloro, bromo, iodo);

each R¹² is independently a substituted alkyl (including lower alkyl),CH₂CN, CH₂N₃, CH₂NH₂, CH₂NHCH₃, CH₂N(CH₃)₂, CH₂OH, halogenated alkyl(including halogenated lower alkyl), CF₃, C(Y³)₃, 2-Br-ethyl, CH₂F,CH₂Cl, CH₂CF₃, CF₂CF₃, C(Y³)₂C(Y³)₃, substituted alkenyl, haloalkenyl(but not Br-vinyl), substituted alkynyl, haloalkynyl, —CH₂C(O)OH,—CH₂C(O)OR⁴, —CH₂C(O)O(lower alkyl), —CH₂C(O)NH₂, —CH₂C(O)NHR⁴,—CH₂C(O)NH(lower alkyl), —CH₂C(O)N(R⁴)₂, —CH₂C(O)N(lower alkyl)₂,—(CH₂)_(m)C(O)OH, —(CH₂)_(m)C(O)OR⁴, —(CH₂)_(m)C(O)O(lower alkyl),—(CH₂)_(m)C(O)NH₂, —(CH₂)_(m)C(O)NHR⁴, —(CH₂)_(m)C(O)NH(lower alkyl),—(CH₂)_(m)C(O)N(R⁴)₂, —(CH₂)_(m)C(O)N(lower alkyl)₂, —C(O)OH, —C(O)OR⁴,—C(O)NH₂, —C(O)NHR⁴, —C(O)NH(lower alkyl), —C(O)N(R⁴)₂, —C(O)N(loweralkyl)₂;

each m is independently 0, 1 or 2;

alternatively, R⁸ and R¹³, R⁹ and R¹³, R⁹ and R¹¹ or R¹⁰ and R¹² cancome together to form a bridged compound selected from the groupconsisting of optionally substituted carbocycle (for example, a 3-7membered carbocyclic ring) or optionally substituted heterocycle (forexample, a 3-7 membered heterocyclic ring having one or more O, S and/orN); or

alternatively, R¹² and R¹³ or R⁹ and R¹⁰ can come together to form aspiro compound selected from the group consisting of optionallysubstituted carbocycle (for example, a 3-7 membered carbocyclic ring) oroptionally substituted heterocycle (for example, a 3-7 memberedheterocyclic ring having one or more O, S and/or N).

In one aspect, R¹ is:

B indicates a spiro compound selected from the group consisting ofoptionally substituted carbocycle (for example, a 3-7 memberedcarbocyclic ring) or optionally substituted heterocycle (for example, a3-7 membered heterocyclic ring having one or more O, S and/or N);

Base is selected from the group consisting of:

wherein each R′, R″, R′″ and R″″ are independently selected from thegroup consisting of H, OH, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, cycloalkyl, Br-vinyl, —O-alkyl, O-alkenyl, O-alkynyl, O-aryl,O-arylalkyl, —O-acyl, O-cycloalkyl, NH₂, NH-alkyl, N-dialkyl, NH-acyl,N-aryl, N-arylalkyl, NH-cycloalkyl, SH, S-alkyl, S-acyl, S-aryl,S-cycloalkyl, S-arylalkyl, F, Cl, Br, I, CN, COOH, CONH₂, CO₂-alkyl,CONH-alkyl, CON-dialkyl, OH, CF₃, CH₂OH, (CH₂)_(m)OH, (CH₂)_(m)NH₂,(CH₂)_(m)COOH, (CH₂)_(m)CN, (CH₂)_(m)NO₂ and (CH₂)_(m)CONH₂;

m is 0 or 1;

each W is independently C—R″ or N;

T and V independently are CH or N;

Q is CH, —CCl, —CBr, —CF, —Cl, —CCN, —C—COOH, —C—CONH₂, or N;

Q₁ and Q₂ independently are N or C—R;

Q₃, Q₄, Q₅ and Q₆ independently are N or CH; and

tautomeric forms thereof.

In another aspect, R¹ is:

G and E independently are selected from the group consisting of CH₃,CH₂OH, CH₂F, CH₂N₃, CH₂CN, (CH₂)_(m)COOH, (CH₂)_(m)COOR, (CH₂)_(m)CONH₂,(CH₂)_(m)CONR₂, (CH₂)_(m)CONHR, N₃ and N-acyl;

m is 0 or 1;

R is H, alkyl or acyl; and

Base is as defined for Formula (XIII).

In one embodiment, at most one of G and E can further be hydrogen.

In another embodiment, R¹ is:

wherein M is selected from the group consisting of O, S, SO, and SO₂;and Base is as defined for Formula (XIII).

In certain embodiments, R¹ is:

wherein A is selected from the group consisting of optionallysubstituted lower alkyl, cycloalkyl, alkenyl, alkynyl, CH₂OH, CH₂NH₂,CH₂NHCH₃, CH₂N(CH₃)₂, CH₂F, CH₂Cl, CH₂N₃, CH₂CN, CH₂CF₃, CF₃, CF₂CF₃,CH₂CO₂R, (CH₂)_(m)COOH, (CH₂)_(m)COOR, (CH₂)_(m)CONH₂, (CH₂)_(m)CONR₂,and (CH₂)_(m)CONHR;

Y is selected from the group consisting of H, optionally substitutedlower alkyl, cycloalkyl, alkenyl, alkynyl, CH₂OH, CH₂NH₂, CH₂NHCH₃,CH₂N(CH₃)₂, CH₂F, CH₂Cl, CH₂N₃, CH₂CN, CH₂CF₃, CF₃, CF₂CF₃, CH₂CO₂R,(CH₂)_(m)COOH, (CH₂)_(m)COOR, (CH₂)_(m)CONH₂, (CH₂)_(m)CONR₂, and(CH₂)_(m)CONHR;

X is selected from the group consisting of H, —OH, optionallysubstituted alkyl, cycloalkyl, alkenyl, alkynyl, —O-alkyl, —O-alkenyl,—O-alkynyl, —O-aryl, —O-arylalkyl, —O-cycloalkyl-, O-acyl, F, Cl, Br, I,CN, NC, SCN, OCN, NCO, NO₂, NH₂, N₃, NH-acyl, NH-alkyl, N-dialkyl,NH-alkenyl, NH-alkynyl, NH-aryl, NH-arylalkyl, NH-cycloalkyl, SH,S-alkyl, S-alkenyl, S-alkynyl, S-aryl, S-arylalkyl, S-acyl,S-cycloalkyl, CO₂-alkyl, CONH-alkyl, CON-dialkyl, CONH-alkenyl,CONH-alkynyl, CONH-arylalkyl, CONH-cycloalkyl, CH₂OH, CH₂NH₂, CH₂NHCH₃,CH₂N(CH₃)₂, CH₂F, CH₂Cl, CH₂N₃, CH₂CN, CH₂CF₃, CF₃, —CF₂CF₃, CH₂CO₂R,(CH₂)_(m)COOH, (CH₂)_(m)COOR, (CH₂)_(m)CONH₂, (CH₂)_(m)CONR₂,(CH₂)_(m)CONHR, an optionally substituted 3-7 membered carbocyclic, andan optionally substituted 3-7 membered heterocyclic ring having O, Sand/or N independently as a heteroatom taken alone or in combination;

m is 0 or 1;

R is H, alkyl or acyl; and Base is a non-natural base selected from thegroup of:

wherein each R′, R″, R′″ and R″″ is independently selected from thegroup consisting of H, OH, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, cycloalkyl, Br-vinyl, —O-alkyl, O-alkenyl, O-alkynyl, O-aryl,O-arylalkyl, —O-acyl, O-cycloalkyl, NH₂, NH-alkyl, N-dialkyl, NH-acyl,N-aryl, N-arylalkyl, NH-cycloalkyl, SH, S-alkyl, S-acyl, S-aryl,S-cycloalkyl, S-arylalkyl, F, Cl, Br, I, CN, COOH, CONH₂, CO₂-alkyl,CONH-alkyl, CON-dialkyl, OH, CF₃, CH₂OH, (CH₂)_(m)OH, (CH₂)_(m)NH₂,(CH₂)_(m)COOH, (CH₂)_(m)CN, (CH₂)_(m)NO₂ and (CH₂)_(m)CONH₂;

m is 0 or 1;

each W is independently C—R″ or N;

T and V independently are CH or N;

Q is CH, —CCl, —CBr, —CF, —Cl, —CCN, —C—COOH, —C—CONH₂, or N;

Q₁ and Q₂ independently are N or C—R″″; and

Q₃, Q₄, Q₅ and Q₆ independently are N or CH;

with the proviso that in bases (g) and (i), R′, R″″ are not H, OH, orNH₂; and Q, T, V, Q₂, Q₅ and Q₆ are not N.

In one embodiment, R¹ is a 2′-(alkyl or aryl) ester or 3′-(alkyl oraryl) ester of 1′, 2′, 3′ or 4′C-branched-β-D or β-L nucleoside with anynatural or non-natural purine or pyrimidine base. In one embodiment, R¹is a 2′ or 3′-(D or L)-amino acid ester of 1′, 2′; 3′ or 4′C-branched-β-D or nucleoside, wherein the amino acid is a natural orsynthetic amino acid. In another embodiment, R¹ is a 3′-D or L-aminoacid ester of 1′, 2′, 3′ or 4′ C-branched-β-D or β-L nucleoside, whereinthe amino acid is a natural or synthetic amino acid. In one embodiment,the amino acid is an L-amino acid.

In one embodiment, the amino acid residue is of the formulaC(O)C(R¹¹)(R¹²)(NR¹³R¹⁴),

wherein R¹¹ is the side chain of an amino acid and wherein, R¹¹ canoptionally be attached to R¹³ to form a ring structure; oralternatively, R¹¹ is an alkyl, aryl, heteroaryl or heterocyclic moiety;

R¹² is hydrogen, alkyl (including lower alkyl) or aryl; and

R¹³ and R¹⁴ are independently hydrogen, acyl (including an acylderivative attached to R¹⁵ or alkyl (including but not limited tomethyl, ethyl, propyl, and cyclopropyl).

In another embodiment, at least one of R² and R³ is an amino acidresidue. In one embodiment, at least one of R² and R³ is L-valinyl.

In one embodiment, R¹ is:

wherein R⁶, R⁷, R⁸, R⁹, R¹⁰ and Base* are as defined in Formula XXX,XXXI, XL, XLI or XLII.

In one embodiment, R¹ is:

wherein R², R³, R⁶ and Base* are as defined in Formula XXX, XXXI, XL,XLI or XLII.

In one embodiment, R¹ is:

wherein X¹ and X² are each independently hydrogen, alkyl, halo or amino;Y is hydrogen, amino, aminoalkyl, aminocycloalkyl, alkyl, cycloalkyl,hydroxy, alkoxy, cycloalkoxy, SH or thioalkyl; X is O or S; and whereinR⁶, R⁷, R⁸, R⁹ are as defined in Formula XXX, XXXI, XL, XLI or XLII.

In one embodiment, R¹ is:

wherein X¹ is hydrogen, alkyl, halo or amino; Y is hydrogen, amino,aminoalkyl, aminocycloalkyl, alkyl, cycloalkyl, hydroxy, alkoxy,cycloalkoxy, SH or thioalkyl; X is O or S; and wherein R⁶, R⁷, R⁸, R⁹are as defined in Formula XXX, XXXI, XL, XLI or XLII.

In one embodiment, R¹ is:

wherein R², R³, and Base* are as defined in Formula XIII, XXX, XXXI, XL,XLI or XLII.

In one embodiment, R¹ is:

wherein R², R³, Y¹, Y³, X¹, and X² are as defined in Formula XIII.

In one embodiment, R¹ is:

wherein R², R³, Y¹, Y³, X¹, and X² are as defined in Formula XIII.

In one embodiment, R¹ is:

wherein R², R³, R⁶, Y, and X¹ are as defined in Formula XIII, XX, XXI orXXII.

In one embodiment, R¹ is:

wherein R², R³, R⁶, R⁷, X and Base* are as defined in Formula XIII, XX,XXI, XXII, XL, XLI or XLII.

In one embodiment, R¹ is

wherein R⁸ is alkyl, alkenyl or alkynyl; R⁷ is OR^(7a);

-   -   R⁹ is OR^(7a);    -   R^(7a) is H or

-   -   R^(m) is a side chain of any natural or non-natural amino acid;        and    -   R^(p) is hydrogen, hydroxy, alkyl or alkoxy; and    -   Base* is as defined in Formula XL, XLI or XLII        In one embodiment, R⁸ is methyl, ethyl, vinyl or ethynyl; R⁷ is        hydroxy or fluoro; R⁹ is hydroxy and other variables are as        described herein.

In one embodiment, R¹ is

In one embodiment, R⁸ is methyl or ethyl. In one embodiment, R^(7a) is Hor

In one embodiment, the phosphoramidate compound provided herein is:

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

In one embodiment, the phosphoramidate compound provided herein is:

In one embodiment, the phosphoramidate compound provided herein is:

In one embodiment, the phosphoramidate compound provided herein is:

In one embodiment, the phosphoramidate compound provided herein is:

In one embodiment, the phosphoramidate compound provided herein is:

In one embodiment, the phosphoramidate compound provided herein is:

In one embodiment, the phosphoramidate compound provided herein is:

In one embodiment, the phosphoramidate compound provided herein is:

In one embodiment, the phosphoramidate compound provided herein is:

In one embodiment, the phosphoramidate compound provided herein is:

In one embodiment, the phosphoramidate compound provided herein is:

In one embodiment, the phosphoramidate compound provided herein is:

In one embodiment, the phosphoramidate compound provided herein is:

In one embodiment, the phosphoramidate compound provided herein is:

In one embodiment, the phosphonoamidate compound provided herein is aphosphonoamidate form of PMPA or PMEA such as:

Optically Active Compounds

It is appreciated that compounds provided herein have several chiralcenters and may exist in and be isolated in optically active and racemicforms. Some compounds may exhibit polymorphism. It is to be understoodthat any racemic, optically-active, diastereomeric, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound provided herein,which possess the useful properties described herein is within the scopeof the invention. It being well known in the art how to prepareoptically active forms (for example, by resolution of the racemic formby recrystallization techniques, by synthesis from optically-activestarting materials, by chiral synthesis, or by chromatographicseparation using a chiral stationary phase).

In particular, since the 1′ and 4′ carbons of a nucleoside are chiral,their nonhydrogen substituents (the base and the CHOR groups,respectively) can be either cis (on the same side) or trans (on oppositesides) with respect to the sugar ring system. The four optical isomerstherefore are represented by the following configurations (whenorienting the sugar moiety in a horizontal plane such that the oxygenatom is in the back): cis (with both groups “up”, which corresponds tothe configuration of naturally occurring β-D nucleosides), cis (withboth groups “down”, which is a nonnaturally occurring β-Lconfiguration), trans (with the C2′ substituent “up” and the C4′substituent “down”), and trans (with the C2′ substituent “down” and theC4′ substituent “up”). The “D-nucleosides” are cis nucleosides in anatural configuration and the “L-nucleosides” are cis nucleosides in thenon-naturally occurring configuration.

Likewise, most amino acids are chiral (designated as L or D, wherein theL enantiomer is the naturally occurring configuration) and can exist asseparate enantiomers.

Examples of methods to obtain optically active materials are known inthe art, and include at least the following.

-   -   i) physical separation of crystals—a technique whereby        macroscopic crystals of the individual enantiomers are manually        separated. This technique can be used if crystals of the        separate enantiomers exist, i.e., the material is a        conglomerate, and the crystals are visually distinct;    -   ii) simultaneous crystallization—a technique whereby the        individual enantiomers are separately crystallized from a        solution of the racemate, possible only if the latter is a        conglomerate in the solid state;    -   iii) enzymatic resolutions—a technique whereby partial or        complete separation of a racemate by virtue of differing rates        of reaction for the enantiomers with an enzyme;    -   iv) enzymatic asymmetric synthesis—a synthetic technique whereby        at least one step of the synthesis uses an enzymatic reaction to        obtain an enantiomerically pure or enriched synthetic precursor        of the desired enantiomer;    -   v) chemical asymmetric synthesis—a synthetic technique whereby        the desired enantiomer is synthesized from an achiral precursor        under conditions that produce asymmetry (i.e., chirality) in the        product, which may be achieved using chiral catalysts or chiral        auxiliaries;    -   vi) diastereomer separations—a technique whereby a racemic        compound is reacted with an enantiomerically pure reagent (the        chiral auxiliary) that converts the individual enantiomers to        diastereomers. The resulting diastereomers are then separated by        chromatography or crystallization by virtue of their now more        distinct structural differences and the chiral auxiliary later        removed to obtain the desired enantiomer;    -   vii) first- and second-order asymmetric transformations—a        technique whereby diastereomers from the racemate equilibrate to        yield a preponderance in solution of the diastereomer from the        desired enantiomer or where preferential crystallization of the        diastereomer from the desired enantiomer perturbs the        equilibrium such that eventually in principle all the material        is converted to the crystalline diastereomer from the desired        enantiomer. The desired enantiomer is then released from the        diastereomer;    -   viii) kinetic resolutions—this technique refers to the        achievement of partial or complete resolution of a racemate (or        of a further resolution of a partially resolved compound) by        virtue of unequal reaction rates of the enantiomers with a        chiral, non-racemic reagent or catalyst under kinetic        conditions;    -   ix) enantiospecific synthesis from non-racemic precursors—a        synthetic technique whereby the desired enantiomer is obtained        from non-chiral starting materials and where the stereochemical        integrity is not or is only minimally compromised over the        course of the synthesis;    -   x) chiral liquid chromatography—a technique whereby the        enantiomers of a racemate are separated in a liquid mobile phase        by virtue of their differing interactions with a stationary        phase. The stationary phase can be made of chiral material or        the mobile phase can contain an additional chiral material to        provoke the differing interactions;    -   xi) chiral gas chromatography—a technique whereby the racemate        is volatilized and enantiomers are separated by virtue of their        differing interactions in the gaseous mobile phase with a column        containing a fixed non-racemic chiral adsorbent phase;    -   xii) extraction with chiral solvents—a technique whereby the        enantiomers are separated by virtue of preferential dissolution        of one enantiomer into a particular chiral solvent;    -   xiii) transport across chiral membranes—a technique whereby a        racemate is placed in contact with a thin membrane barrier. The        barrier typically separates two miscible fluids, one containing        the racemate, and a driving force such as concentration or        pressure differential causes preferential transport across the        membrane barrier. Separation occurs as a result of the        non-racemic chiral nature of the membrane which allows only one        enantiomer of the racemate to pass through.

In some embodiments, compositions of phosphonoamidate or phosphoramidatecompounds are provided that are substantially free of a designatedenantiomer of that nucleoside. In a preferred embodiment, in the methodsand compounds of this invention, the compounds are substantially free ofenantiomers. In some embodiments, the composition includes that includesa compound that is at least 85, 90%, 95%, 98%, 99% to 100% by weight, ofthe compound, the remainder comprising other chemical species orenantiomers.

Preparation of Compounds

The compounds provided herein can be prepared, isolated or obtained byany method apparent to those of skill in the art. Exemplary methods ofpreparation are described in detail in the examples below.

In certain embodiments, compounds provided herein can be prepared bycoupling alcohols and H-phosphonate monoesters as illustrated in thereaction scheme below:

any reactive function on R^(y), R⁷, R⁸, R⁹, R¹⁰ or on the base may beprotected during the coupling reaction. A variety of coupling agentsknown to one of skill in the art can be used. Exemplary coupling agentsfor use in the reaction include, but are not limited to HOBt(N-Hydroxybenzotriazole), HBTU(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate), DCC(N,N′-dicyclohexylcarbodiimide), BOP(Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniurnhexafluorophosphate),PyBOP (1H-benzotriazol-1-yloxytripyrrolidinophosphoniumhexafluorophosphate) and others known to one of skill in the art.

A general scheme for the synthesis of hydroxytBuSATEN-benzylphosphoramidate nucleoside derivatives represented by B isprovided in Schemes B1-B3 below.

where R═H, Tr, MMTr or DMTr in case of reactive amine; R¹, R², R⁴, R⁶═H,alkyl or halo and R³/R⁵ are both H or isopropylidene.

In addition, certain nucleosides and analogs thereof and prodrugsthereof can be prepared according to methods described in U.S. Pat. Nos.6,812,219; 7,105,493; 7,101,861; 6,914,054; 6,555,676; 7,202,224;7,105,499; 6,777,395; 6,914,054; 7,192,936; US publication Nos.2005203243; 2007087960; 2007060541; 2007060505; 2007060504; 2007060503;2007060498; 2007042991; 2007042990; 2007042940; 2007042939 and2007037735; International Publication Nos. WO 04/003000; WO 04/022999;WO 04/002422; WO 01/90121 and WO 01/92282. Other patents/patentapplications disclosing nucleoside analogs to treat hepatitis C virusthat can be derivatized as described herein include: PCT/CA00/01316 (WO01/32153; filed Nov. 3, 2000) and PCT/CA01/00197 (WO 01/60315; filedFeb. 19, 2001) filed by BioChem Pharma, Inc. (now Shire Biochem, Inc.);PCT/US02/01531 (WO 02/057425; filed Jan. 18, 2002); PCT/US02/03086 (WO02/057287; filed Jan. 18, 2002); U.S. Pat. No. 7,202,224; U.S. Pat. No.7,125,855; U.S. Pat. No. 7,105,499 and U.S. Pat. No. 6,777,395 by Merck& Co., Inc.; PCT/EP01/09633 (WO 02/18404; published Aug. 21, 2001); US2006/0040890; 2005/0038240; 2004/0121980; U.S. Pat. Nos. 6,846,810;6,784,166 and 6,660,721 by Roche; PCT Publication Nos. WO 01/79246(filed Apr. 13, 2001), WO 02/32920 (filed Oct. 18, 2001) and WO02/48165; US 2005/0009737 and US 2005/0009737; U.S. Pat. Nos. 7,094,770and 6,927,291 by Pharmasset, Ltd. Contents of these references arehereby incorporated by reference in their entireties.

Assay Methods

Compounds can be assayed for HBV activity according to any assay knownto those of skill in the art. Compounds can be assayed for HCV activityaccording to any assay known to those of skill in the art.

Further, compounds can be assayed for accumulation in liver cells of asubject according to any assay known to those of skill in the art. Incertain embodiments, a compound can be administered to the subject, anda liver cell of the subject can be assayed for the compound or aderivative thereof, e.g. a nucleoside, nucleoside phosphate ornucleoside triphosphate derivative thereof.

In one embodiment, a phosphoramidate or phosphonoamidate nucleosidecompound is administered to cells, such as liver cells, in vivo or invitro, and the nucleoside triphosphate levels delivered intracellularlyare measured, to indicate delivery of the compound andtriphosphorylation in the cell. The levels of intracellular nucleosidetriphosphate can be measured using analytical techniques known in theart. Methods of detecting ddATP are described herein below by way ofexample, but other nucleoside triphosphates can be readily detectedusing the appropriate controls, calibration samples and assaytechniques.

In one embodiment, ddATP concentrations are measured in a sample bycomparison to calibration standards made from control samples. The ddATPconcentrations in a sample can be measured using an analytical methodsuch as HPLC LC MS. In one embodiment, a test sample is compared to acalibration curve created with known concentrations of ddATP to therebyobtain the concentration of that sample.

In one embodiment, the samples are manipulated to remove impurities suchas salts (Na⁺, K⁺, etc.) before analysis. In one embodiment, the lowerlimit of quantitation is about ˜0.2 pmol/mL for hepatocyte cellularextracts particularly where reduced salt is present.

In one embodiment, the method allows successfully measuring triphosphatenucleotides formed at levels of 1-10,000 pmol per million cells in e.g.cultured hepatocytes and HepG2 cells.

Methods of Use

The phosphoramidate and phosphonoamidate compounds of a variety oftherapeutic agents can be formed using methods available in the art andthose disclosed herein. Such compounds can be used in some embodimentsto enhance delivery of the drug to the liver.

In one embodiment, the compound comprises a S-acyl-2-thioethylphosphoramidate or S-acyl-2-thioethyl phosphonoamidate, e.g., aS-pivaloyl-2-thioethyl phosphoramidate or S-hydroxypivaloyl-2-thioethylphosphonoamidate derivative. Therapeutic agents that can be derivatizedto phosphoramidate or phosphonoamidate compound form include anyanti-viral agent that includes, or has been derivatized to include areactive group for attachment of the phosphoramidate or phosphonoamidatemoiety, including but not limited to nucleosides and nucleosideanalogues including acyclic nucleosides.

Advantageously, such phosphoramidate and phosphonamidate compoundsadvantageously can have enhanced delivery to the liver. In someembodiments, the compounds permit delivery of an active 5′-monophosphateof a nucleoside to the liver, which can enhance the formation of activetriphosphorylated compound.

In one embodiment, provided herein are methods for the treatment and/orprophylaxis of a host infected with Flaviviridae that includes theadministration of an effective amount of a compounds provided herein, ora pharmaceutically acceptable salt thereof. In one embodiment, providedherein are methods for treating an HCV infection in a subject. Incertain embodiments, the methods encompass the step of administering tothe subject in need thereof an amount of a compound effective for thetreatment or prevention of an HCV infection in combination with a secondagent effective for the treatment or prevention of the infection. Thecompound can be any compound as described herein, and the second agentcan be any second agent described in the art or herein. In certainembodiments, the compound is in the form of a pharmaceutical compositionor dosage form, as described in the sections above.

Flaviviridae that can be treated are discussed generally in FieldsVirology, Editors: Fields, B. N., Knipe, D. M., and Howley, P. M.,Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 31, 1996. In aparticular embodiment of the invention, the Flaviviridae is HCV. In analternate embodiment of the invention, the Flaviviridae is a flavivirusor pestivirus. Specific flaviviruses include, without limitation:Absettarov, Alfuy, Apoi, Aroa, Bagaza, Banzi, Bouboui, Bussuquara,Cacipacore, Carey Island, Dakar bat, Dengue 1, Dengue 2, Dengue 3,Dengue 4, Edge Hill, Entebbe bat, Gadgets Gully, Hanzalova, Hypr,Ilheus, Israel turkey meningoencephalitis, Japanese encephalitis, Jugra,Jutiapa, Kadam, Karshi, Kedougou, Kokobera, Koutango, Kumlinge, Kunjin,Kyasanur Forest disease, Langat, Louping ill, Meaban, Modoc, Montanamyotis leukoencephalitis, Murray valley encephalitis, Naranjal, Negishi,Ntaya, Omsk hemorrhagic fever, Phnom-Penh bat, Powassan, Rio Bravo,Rocio, Royal Farm, Russian spring-summer encephalitis, Saboya, St. Louisencephalitis, Sal Vieja, San Perlita, Saumarez Reef, Sepik, Sokuluk,Spondweni, Stratford, Tembusu, Tyuleniy, Uganda S, Usutu, Wesselsbron,West Nile, Yaounde, Yellow fever, and Zika.

Pestiviruses that can be treated are discussed generally in FieldsVirology, Editors: Fields, B. N., Knipe, D. M., and Howley, P. M.,Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 33, 1996.Specific pestiviruses include, without limitation: bovine viral diarrheavirus (“BVDV”), classical swine fever virus (“CSFV,” also called hogcholera virus), and border disease virus (“BDV”).

In certain embodiments, provided herein are methods for the treatmentand/or prophylaxis of hepatitis B infections that includes administeringan effective amount of a compound as described herein, e.g. of FormulaI, IIa or IIb, its pharmaceutically acceptable salt or composition. Inanother embodiment, provided herein are methods of treatmentand/prophylaxis of conditions related to hepatitis B infections, such asanti-HBV antibody positive and HBV-positive conditions, chronic liverinflammation caused by HBV, cirrhosis, acute hepatitis, fulminanthepatitis, chronic persistent hepatitis, and fatigue. In certainembodiments, provided herein are prophylactic methods to prevent orretard the progression of clinical illness in individuals who areanti-HBV antibody or HBV-antigen positive or who have been exposed toHBV.

In certain embodiments, the subject can be any subject infected with, orat risk for infection with, HCV and/or HBV. Infection or risk forinfection can be determined according to any technique deemed suitableby the practitioner of skill in the art. In one embodiment, subjects arehumans infected with HCV and/or HBV.

In certain embodiments, the subject has never received therapy orprophylaxis for an HCV and/or HBV infection. In further embodiments, thesubject has previously received therapy or prophylaxis for an HCV and/orHBV infection. For instance, in certain embodiments, the subject has notresponded to an HCV and/or HBV therapy. For example, under currentinterferon therapy, up to 50% or more HCV subjects do not respond totherapy. In certain embodiments, the subject can be a subject thatreceived therapy but continued to suffer from viral infection or one ormore symptoms thereof. In certain embodiments, the subject can be asubject that received therapy but failed to achieve a sustainedvirologic response. In certain embodiments, the subject has receivedtherapy for an HCV and/or HBV infection but has failed to show, forexample, a 2 log₁₀ decline in HCV RNA levels after 12 weeks of therapy.It is believed that subjects who have not shown more than 2 log₁₀reduction in serum HCV RNA after 12 weeks of therapy have a 97-100%chance of not responding.

In certain embodiments, the subject is a subject that discontinued anHCV and/or HBV therapy because of one or more adverse events associatedwith the therapy. In certain embodiments, the subject is a subject wherecurrent therapy is not indicated. For instance, certain therapies forHCV are associated with neuropsychiatric events. Interferon (IFN)-alfaplus ribavirin is associated with a high rate of depression. Depressivesymptoms have been linked to a worse outcome in a number of medicaldisorders. Life-threatening or fatal neuropsychiatric events, includingsuicide, suicidal and homicidal ideation, depression, relapse of drugaddiction/overdose, and aggressive behavior have occurred in subjectswith and without a previous psychiatric disorder during HCV therapy.Interferon-induced depression is a limitation for the treatment ofchronic hepatitis C, especially for subjects with psychiatric disorders.Psychiatric side effects are common with interferon therapy andresponsible for about 10% to 20% of discontinuations of current therapyfor HCV infection.

Accordingly, provided are methods of treating or preventing an HCVinfection in subjects where the risk of neuropsychiatric events, such asdepression, contraindicates treatment with current HCV therapy. In oneembodiment, provided are methods of treating or preventing HCV infectionin subjects where a neuropsychiatric event, such as depression, or riskof such indicates discontinuation of treatment with current HCV therapy.Further provided are methods of treating or preventing HCV infection insubjects where a neuropsychiatric event, such as depression, or risk ofsuch indicates dose reduction of current HCV therapy.

Current therapy is also contraindicated in subjects that arehypersensitive to interferon or ribavirin, or both, or any othercomponent of a pharmaceutical product for administration of interferonor ribavirin. Current therapy is not indicated in subjects withhemoglobinopathies (e.g., thalassemia major, sickle-cell anemia) andother subjects at risk from the hematologic side effects of currenttherapy. Common hematolygic side effects include bone marrowsuppression, neutropenia and thrombocytopenia. Furthermore, ribavirin istoxic to red blood cells and is associated with hemolysis. Accordingly,in one embodiment, provided are methods of treating or preventing HCVinfection in subjects hypersensitive to interferon or ribavirin, orboth, subjects with a hemoglobinopathy, for instance thalassemia majorsubjects and sickle-cell anemia subjects, and other subjects at riskfrom the hematologic side effects of current therapy.

In certain embodiments, the subject has received an HCV and/or HBVtherapy and discontinued that therapy prior to administration of amethod provided herein. In further embodiments, the subject has receivedtherapy and continues to receive that therapy along with administrationof a method provided herein. The methods can be co-administered withother therapy for HBC and/or HCV according to the judgment of one ofskill in the art. In certain embodiments, the methods or compositionsprovided herein can be co-administered with a reduced dose of the othertherapy for HBC and/or HCV.

In certain embodiments, provided are methods of treating a subject thatis refractory to treatment with interferon. For instance, in someembodiments, the subject can be a subject that has failed to respond totreatment with one or more agents selected from the group consisting ofinterferon, interferon α, pegylated interferon α, interferon plusribavirin, interferon α plus ribavirin and pegylated interferon α plusribavirin. In some embodiments, the subject can be a subject that hasresponded poorly to treatment with pne or more agents selected from thegroup consisting of interferon, interferon α, pegylated interferon α,interferon plus ribavirin, interferon α plus ribavirin and pegylatedinterferon α plus ribavirin. A pro-drug form of ribavirin, such astaribavirin, may also be used.

In certain embodiments, the subject has, or is at risk for, co-infectionof HCV with HIV. For instance, in the United States, 30% of HIV subjectsare co-infected with HCV and evidence indicates that people infectedwith HIV have a much more rapid course of their hepatitis C infection.Maier and Wu, 2002, World J Gastroenterol 8:577-57. The methods providedherein can be used to treat or prevent HCV infection in such subjects.It is believed that elimination of HCV in these subjects will lowermortality due to end-stage liver disease. Indeed, the risk ofprogressive liver disease is higher in subjects with severeAIDS-defining immunodeficiency than in those without. See, e.g., Lesenset al., 1999, J Infect Dis 179:1254-1258. In one embodiment, compoundsprovided herein have been shown to suppress HIV in HIV subjects. Thus,in certain embodiments, provided are methods of treating or preventingHIV infection and HCV infection in subjects in need thereof.

In certain embodiments, the compounds or compositions are administeredto a subject following liver transplant. Hepatitis C is a leading causeof liver transplantation in the U.S. and many subjects that undergoliver transplantation remain HCV positive following transplantation. Inone embodiment, provided are methods of treating such recurrent HCVsubjects with a compound or composition provided herein. In certainembodiments, provided are methods of treating a subject before, duringor following liver transplant to prevent recurrent HCV infection.

In certain embodiments, provided herein are methods for the treatmentand/or prophylaxis of hepatitis B infections and other relatedconditions such as anti-HBV antibody positive and HBV-positiveconditions, chronic liver inflammation caused by HBV, cirrhosis, acutehepatitis, fulminant hepatitis, chronic persistent hepatitis, andfatigue that includes administering an effective amount of a compound orcomposition provided herein.

In one embodiment, provided herein are methods for treatment and/orprophylaxis of hepatitis B infections and other related conditions suchas anti-HBV antibody positive and HBV-positive conditions, chronic liverinflammation caused by HBV, cirrhosis, acute hepatitis, fulminanthepatitis, chronic persistent hepatitis, and fatigue that includesadministering an effective amount of a compound or composition providedherein.

Second Therapeutic Agents

In certain embodiments, the compounds and compositions provided hereinare useful in methods of treatment of a liver disorder, that comprisesfurther administration of a second agent effective for the treatment ofthe disorder, such as HCV and/or HBV infection in a subject in needthereof. The second agent can be any agent known to those of skill inthe art to be effective for the treatment of the disorder, includingthose currently approved by the FDA.

In certain embodiments, a compound provided herein is administered incombination with one second agent. In further embodiments, a secondagent is administered in combination with two second agents. In stillfurther embodiments, a second agent is administered in combination withtwo or more second agents.

As used herein, the term “in combination” includes the use of more thanone therapy (e.g., one or more prophylactic and/or therapeutic agents).The use of the term “in combination” does not restrict the order inwhich therapies (e.g., prophylactic and/or therapeutic agents) areadministered to a subject with a disorder. A first therapy (e.g., aprophylactic or therapeutic agent such as a compound provided herein)can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes,45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequentto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks after) the administration of a second therapy (e.g., aprophylactic or therapeutic agent) to a subject with a disorder.

As used herein, the term “synergistic” includes a combination of acompound provided herein and another therapy (e.g., a prophylactic ortherapeutic agent) which has been or is currently being used to prevent,manage or treat a disorder, which is more effective than the additiveeffects of the therapies. A synergistic effect of a combination oftherapies (e.g., a combination of prophylactic or therapeutic agents)permits the use of lower dosages of one or more of the therapies and/orless frequent administration of said therapies to a subject with adisorder. The ability to utilize lower dosages of a therapy (e.g., aprophylactic or therapeutic agent) and/or to administer said therapyless frequently reduces the toxicity associated with the administrationof said therapy to a subject without reducing the efficacy of saidtherapy in the prevention or treatment of a disorder). In addition, asynergistic effect can result in improved efficacy of agents in theprevention or treatment of a disorder. Finally, a synergistic effect ofa combination of therapies (e.g., a combination of prophylactic ortherapeutic agents) may avoid or reduce adverse or unwanted side effectsassociated with the use of either therapy alone.

The active compounds provided herein can be administered in combinationor alternation with another therapeutic agent, in particular an anti-HCVor hepatitis B agent. In combination therapy, effective dosages of twoor more agents are administered together, whereas in alternation orsequential-step therapy, an effective dosage of each agent isadministered serially or sequentially. The dosages given will depend onabsorption, inactivation and excretion rates of the drug as well asother factors known to those of skill in the art. It is to be noted thatdosage values will also vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens and schedules should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions. In certain embodiments, an anti-HCV (or anti-pestivirus oranti-flavivirus) compound that exhibits an EC₅₀ of 10-15 μM, orpreferably less than 1-5 μM, is desirable.

It has been recognized that drug-resistant variants of flaviviruses,pestiviruses or HCV can emerge after prolonged treatment with anantiviral agent. Drug resistance most typically occurs by mutation of agene that encodes for an enzyme used in viral replication. The efficacyof a drug against the viral infection can be prolonged, augmented, orrestored by administering the compound in combination or alternationwith a second, and perhaps third, antiviral compound that induces adifferent mutation from that caused by the principle drug.Alternatively, the pharmacokinetics, biodistribution or other parameterof the drug can be altered by such combination or alternation therapy.In general, combination therapy is typically preferred over alternationtherapy because it induces multiple simultaneous stresses on the virus.

Any of the viral treatments described in the Background of the Inventioncan be used in combination or alternation with the compounds describedin this specification. Nonlimiting examples of second agents include:

HCV Protease inhibitors: Examples include Medivir HCV Protease Inhibitor(Medivir/Tobotec); ITMN-191 (InterMune), SCH 503034 (Schering) and VX950(Vertex). Further examples of protease inhibitors includesubstrate-based NS3 protease inhibitors (Attwood et al., Antiviralpeptide derivatives, PCT WO 98/22496, 1998; Attwood et al., AntiviralChemistry and Chemotherapy 1999, 10, 259-273; Attwood et al.,Preparation and use of amino acid derivatives as anti-viral agents,German Patent Pub. DE 19914474; Tung et al. Inhibitors of serineproteases, particularly hepatitis C virus NS3 protease, PCT WO98/17679), including alphaketoamides and hydrazinoureas, and inhibitorsthat terminate in an electrophile such as a boronic acid or phosphonate(Llinas-Brunet et al, Hepatitis C inhibitor peptide analogues, PCT WO99/07734); Non-substrate-based NS3 protease inhibitors such as2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al.,Biochemical and Biophysical Research Communications, 1997, 238, 643-647;Sudo K. et al. Antiviral Chemistry and Chemotherapy, 1998, 9, 186),including RD3-4082 and RD3-4078, the former substituted on the amidewith a 14 carbon chain and the latter processing a para-phenoxyphenylgroup; and Sch 68631, a phenanthrenequinone, an HCV protease inhibitor(Chu M. et al., Tetrahedron Letters 37:7229-7232, 1996).

SCH 351633, isolated from the fungus Penicillium griseofulvum, wasidentified as a protease inhibitor (Chu M. et al., Bioorganic andMedicinal Chemistry Letters 9:1949-1952). Eglin c, isolated from leech,is a potent inhibitor of several serine proteases such as S. griseusproteases A and B, α-chymotrypsin, chymase and subtilisin. Qasim M. A.et al., Biochemistry 36:1598-1607, 1997.

U.S. patents disclosing protease inhibitors for the treatment of HCVinclude, for example, U.S. Pat. No. 6,004,933 to Spruce et al. whichdiscloses a class of cysteine protease inhibitors for inhibiting HCVendopeptidase 2; U.S. Pat. No. 5,990,276 to Zhang et al. which disclosessynthetic inhibitors of hepatitis C virus NS3 protease; U.S. Pat. No.5,538,865 to Reyes et a; WO 02/008251 to Corvas International, Inc, andU.S. Pat. No. 7,169,760, US2005/176648, WO 02/08187 and WO 02/008256 toSchering Corporation. HCV inhibitor tripeptides are disclosed in U.S.Pat. Nos. 6,534,523, 6,410,531, and 6,420,380 to Boehringer Ingelheimand WO 02/060926 to Bristol Myers Squibb. Diaryl peptides as NS3 serineprotease inhibitors of HCV are disclosed in WO 02/48172 and U.S. Pat.No. 6,911,428 to Schering Corporation. Imidazoleidinones as NS3 serineprotease inhibitors of HCV are disclosed in WO 02/08198 and U.S. Pat.No. 6,838,475 to Schering Corporation and WO 02/48157 and U.S. Pat. No.6,727,366 to Bristol Myers Squibb. WO 98/17679 and U.S. Pat. No.6,265,380 to Vertex Pharmaceuticals and WO 02/48116 and U.S. Pat. No.6,653,295 to Bristol Myers Squibb also disclose HCV protease inhibitors.Further examples of HCV serine protease inhibitors are provided in U.S.Pat. No. 6,872,805 (Bristol-Myers Squibb); WO 2006000085 (BoehringerIngelheim); U.S. Pat. No. 7,208,600 (Vertex); US 2006/0046956(Schering-Plough); WO 2007/001406 (Chiron); US 2005/0153877; WO2006/119061 (Merck); WO 00/09543 (Boehringer Ingelheim), U.S. Pat. No.6,323,180 (Boehringer Ingelheim) WO 03/064456 (Boehringer Ingelheim),U.S. Pat. No. 6,642,204 (Boehringer Ingelheim), WO 03/064416 (BoehringerIngelheim), U.S. Pat. No. 7,091,184 (Boehringer Ingelheim), WO 03/053349(Bristol-Myers Squibb), U.S. Pat. No. 6,867,185, WO 03/099316(Bristol-Myers Squibb), U.S. Pat. No. 6,869,964, WO 03/099274(Bristol-Myers Squibb), U.S. Pat. No. 6,995,174, WO 2004/032827(Bristol-Myers Squibb), U.S. Pat. No. 7,041,698, WO 2004/043339 and U.S.Pat. No. 6,878,722 (Bristol-Myers Squibb).

Thiazolidine derivatives which show relevant inhibition in areverse-phase HPLC assay with an NS3/4A fusion protein and NS5A/5Bsubstrate (Sudo K. et at, Antiviral Research, 1996, 32, 9-18),especially compound RD-1-6250, possessing a fused cinnamoyl moietysubstituted with a long alkyl chain, RD4 6205 and RD4 6193;

Thiazolidines and benzanilides identified in Kakiuchi N. et al. J. EBSLetters 421, 217-220; Takeshita N. et al. Analytical Biochemistry, 1997,247, 242-246;

A phenanthrenequinone possessing activity against protease in a SDS-PAGEand autoradiography assay isolated from the fermentation culture brothof Streptomyces sp., SCH 68631 (Chu M. et al., Tetrahedron Letters,1996, 37, 7229-7232), and SCH 351633, isolated from the fungusPenicillium griseofulvum, which demonstrates activity in a scintillationproximity assay (Chu M. et al., Bioorganic and Medicinal ChemistryLetters 9, 1949-1952);

Helicase inhibitors (Diana G. D. et al., Compounds, compositions andmethods for treatment of hepatitis C, U.S. Pat. No. 5,633,358; Diana G.D. et al., Piperidine derivatives, pharmaceutical compositions thereofand their use in the treatment of hepatitis C, PCT WO 97/36554);

Nucleotide polymerase inhibitors and gliotoxin (Ferrari R. et al.Journal of Virology, 1999, 73, 1649-1654), and the natural productcerulenin (Lohmann V. et al., Virology, 1998, 249, 108-118);

Interfering RNA (iRNA) based antivirals, including short interfering RNA(siRNA) based antivirals, such as Sima-034 and others described inInternational Patent Publication Nos. WO/03/070750 and WO 2005/012525,and US Patent Publication No. US 2004/0209831.

Antisense phosphorothioate oligodeoxynucleotides (S-ODN) complementaryto sequence stretches in the 5′ non-coding region (NCR) of the virus(Alt M. et al., Hepatology, 1995, 22, 707-717), or nucleotides 326-348comprising the 3′ end of the NCR and nucleotides 371-388 located in thecore coding region of the HCV RNA (Alt M. et al., Archives of Virology,1997, 142, 589-599; Galderisi U. et al., Journal of Cellular Physiology,1999, 181, 251-257);

Inhibitors of IRES-dependent translation (Ikeda N et al., Agent for theprevention and treatment of hepatitis C, Japanese Patent Pub.JP-08268890; Kai Y. et al. Prevention and treatment of viral diseases,Japanese Patent Pub. JP-10101591);

Ribozymes, such as nuclease-resistant ribozymes (Maccjak, D. J. et al.,Hepatology 1999, 30, abstract 995) and those disclosed in U.S. Pat. No.6,043,077 to Barber et al., and U.S. Pat. Nos. 5,869,253 and 5,610,054to Draper et al.; and

Nucleoside analogs have also been developed for the treatment ofFlaviviridae infections.

In certain embodiments, the compounds provided herein can beadministered in combination with any of the compounds described byIdenix Pharmaceuticals in International Publication Nos. WO 01/90121, WO01/92282, WO 2004/003000, 2004/002422 and WO 2004/002999.

Other patent applications disclosing the use of certain nucleosideanalogs that can be used as second agents to treat hepatitis C virusinclude: PCT/CA00/01316 (WO 01/32153; filed Nov. 3, 2000) andPCT/CA01/00197 (WO 01/60315; filed Feb. 19, 2001) filed by BioChemPharma, Inc. (now Shire Biochem, Inc.); PCT/US02/01531 (WO 02/057425;filed Jan. 18, 2002); PCT/US02/03086 (WO 02/057287; filed Jan. 18,2002); U.S. Pat. Nos. 7,202,224; 7,125,855; 7,105,499 and 6,777,395 byMerck & Co., Inc.; PCT/EP01/09633 (WO 02/18404; published Aug. 21,2001); US 2006/0040890; 2005/0038240; 2004/0121980; 6,846,810; 6,784,166and 6,660,721 by Roche; PCT Publication Nos. WO 01/79246 (filed Apr. 13,2001), WO 02/32920 (filed Oct. 18, 2001) and WO 02/48165; US2005/0009737; US 2005/0009737; 7,094,770 and 6,927,291 by Pharmasset,Ltd.

Further compounds that can be used as second agents to treat hepatitis Cvirus are disclosed in PCT Publication No. WO 99/43691 to EmoryUniversity, entitled “2′-Fluoronucleosides”. The use of certain2′-fluoronucleosides to treat HCV is disclosed.

Other miscellaneous compounds that can be used as second agents include1-amino-alkylcyclohexanes (U.S. Pat. No. 6,034,134 to Gold et al.),alkyl lipids (U.S. Pat. No. 5,922,757 to Chojkier et al.), vitamin E andother antioxidants (U.S. Pat. No. 5,922,757 to Chojkier et al.),squalene, amantadine, bile acids (U.S. Pat. No. 5,846,964 to Ozeki etal.), N-(phosphonoacetyl)-L-aspartic acid, (U.S. Pat. No. 5,830,905 toDiana et al.), benzenedicarboxamides (U.S. Pat. No. 5,633,388 to Dianaet al.), polyadenylic acid derivatives (U.S. Pat. No. 5,496,546 to Wanget al.), 2′,3′-dideoxyinosine (U.S. Pat. No. 5,026,687 to Yarchoan etal.), benzimidazoles (U.S. Pat. No. 5,891,874 to Colacino et al.), plantextracts (U.S. Pat. No. 5,837,257 to Tsai et al., U.S. Pat. No.5,725,859 to Omer et al., and U.S. Pat. No. 6,056,961), and piperidenes(U.S. Pat. No. 5,830,905 to Diana et al.).

Exemplary Second Agents for Treatment of HCV

In one embodiment, one or more compounds provided herein can beadministered in combination or alternation with an anti-hepatitis Cvirus interferon, such as Intron A® (interferon alfa-2b) and Pegasys®(Peginterferon alfa-2a); Roferon A® (Recombinant interferon alfa-2a),Infergen® (consensus interferon; interferon alfacon-1), PEG-Intron®(pegylated interferon alfa-2b) and Pegasys® (pegylated interferonalfa-2a).

In one embodiment, the anti-hepatitis C virus interferon is infergen,IL-29 (PEG-Interferon lambda), 87025 (Maxy-alpha), Belerofon, OralInterferon alpha, BLX-883 (Locteron), omega interferon, multiferon,medusa interferon, Albuferon or REBIF®.

In one embodiment, one or more compounds provided herein can beadministered in combination or alternation with an anti-hepatitis Cvirus polymerase inhibitor, such as ribavirin, viramidine, NM 283(valopicitabine), PSI-6130, R1626, HCV-796 or R7128.

In certain embodiments, the one or more compounds provided herein can beadministered in combination with ribavarin and an anti-hepatitis C virusinterferon, such as Intron A® (interferon alfa-2b) and Pegasys®(Peginterferon alfa-2a); Roferon A® (Recombinant interferon alfa-2a),Infergen® (consensus interferon; interferon alfacon-1), PEG-Intron®(pegylated interferon alfa-2b) and Pegasys® (pegylated interferonalfa-2a).

In one embodiment, one or more compounds provided herein can beadministered in combination or alternation with an anti-hepatitis Cvirus protease inhibitor such as ITMN-191, SCH 503034, VX950(telaprevir) or Medivir HCV Protease Inhibitor.

In one embodiment, one or more compounds provided herein can beadministered in combination or alternation with an anti-hepatitis Cvirus vaccine, such as TG4040, PeviPRO™, CGI-5005, HCV/MF59, GV1001,IC41 or INN00101 (E1).

In one embodiment, one or more compounds provided herein can beadministered in combination or alternation with an anti-hepatitis Cvirus monoclonal antibody, such as AB68 or XTL-6865 (formerly HepX-C);or an anti-hepatitis C virus polyclonal antibody, such as cicavir.

In one embodiment, one or more compounds provided herein can beadministered in combination or alternation with an anti-hepatitis Cvirus immunomodulator, such as Zadaxin® (thymalfasin), NOV-205 orOglufanide.

In one embodiment, one or more compounds provided herein can beadministered in combination or alternation with Nexavar, doxorubicin,PI-88, amantadine, JBK-122, VGX-410C, MX-3253 (Ceglosivir), Suvus(BIVN-401 or virostat), PF-03491390 (formerly IDN-6556), G126270,UT-231B, DEBIO-025, EMZ702, ACH-0137171, MitoQ, ANA975, AVI-4065,Bavituxinab (Tarvacin), Alinia (nitrazoxanide) or PYN17.

Exemplary Second Agents for Treatment of HBV

It has been recognized that drug-resistant variants of HBV can emergeafter prolonged treatment with an antiviral agent. Drug resistance mosttypically occurs by mutation of a gene that encodes for an enzyme usedin the viral life cycle, and most typically in the case of HBV, DNApolymerase. The efficacy of a drug against HBV infection can beprolonged, augmented, or restored by administering the compound incombination or alternation with a second, and perhaps third, antiviralcompound that induces a different mutation from that caused by theprinciple drug. Alternatively, the pharmacokinetics, biodistribution, orother parameter of the drug can be altered by such combination oralternation therapy. In general, combination therapy is typicallypreferred over alternation therapy because it induces multiplesimultaneous stresses on the virus.

The anti-hepatitis B viral activity of compounds provided herein can beenhanced by administering one or more of these further agents incombination or alternation. Alternatively, for example, one or morecompounds provided herein can be administered in combination oralternation with any other known anti-hepatitis B virus agent. Suchagents include anti-hepatitis B virus interferons, such as Intron A®(interferon alfa-2b) and Pegasys® (Peginterferon alfa-2a); polymeraseinhibitors, such as Epivir-HBV (lamivudine), Hepsera (adefovirdipivoxil), baraclude (entecavir), Tyzeka (telbivudine), Emtricitabine(FTC), Clevudine (L-FMAU), Viread (tenofovir), Valtorcitabine,Amdoxovir, ANA 380, Pradefovir (remofovir) and RCV (racivir); vaccines,such as Hi-8 HBV, HepaVaxx B and HBV Core Antigen vaccine; and otheragents, such as HepX, SpecifEx-HepB, Zadaxin, EHT899, Bay 41-4109, UT231-B, HepeX-B and NOV-205 or any other compound that exhibits an EC₅₀of less than 15 micromolar in 2.2.15 cells; or their prodrugs orpharmaceutically acceptable salts. Several other examples of anti-HBVagents are provided in U.S. Application Publication No. 20050080034 andinternational publication no. WO 2004/096286, which are incorporated byreference in their entireties.

In one embodiment, one or more compounds provided herein can beadministered in combination or alternation with anti-hepatitis B virusagent such as interferon α-2b, peginterferon α-2a, lamivudine, hepsera,baraclude, telbivudine, emtricitabine, clevudine, tenofovir,valtorcitabine, amdoxovir, ANA 380, remofovir, racivir, alinia, Hi-8 HBVand HepaVaxx B.

In another embodiment, a compound provided herein is administered incombination or alternation with an immune modulator or otherpharmaceutically active modifier of viral replication, including abiological material such as a protein, peptide, oligonucleotide, orgamma globulin, including but not limited to interfereon, interleukin,or an antisense oligonucleotides to genes which express or regulatehepatitis B replication.

Any method of alternation can be used that provides treatment to thepatient. Nonlimiting examples of alternation patterns include 1-6 weeksof administration of an effective amount of one agent followed by 1-6weeks of administration of an effective amount of a second anti-HBVagent. The alternation schedule can include periods of no treatment.Combination therapy generally includes the simultaneous administrationof an effective ratio of dosages of two or more anti-HBV agents.

In light of the fact that HBV is often found in patients who are alsoanti-HIV antibody or HIV-antigen positive or who have been exposed toHIV, the active anti-HBV compounds disclosed herein or their derivativesor prodrugs can be administered in the appropriate circumstance incombination or alternation with anti-HIV medications.

The compounds provided herein can also be administered in combinationwith antibiotics, other antiviral compounds, antifungal agents or otherpharmaceutical agents administered for the treatment of secondaryinfections.

Pharmaceutical Compositions and Methods of Administration

Phosphoramidate and phosphonoamidate compounds of a variety oftherapeutic agents can be formulated into pharmaceutical compositionsusing methods available in the art and those disclosed herein. Suchcompounds can be used in some embodiments to enhance delivery of thedrug to the liver. In one embodiment, the compound comprises aS-acyl-2-thioethyl phosphoramidate or S-acyl-2-thioethylphosphonoamidate, e.g., a S-pivaloyl-2-thioethyl phosphoramidate orS-hydroxypivaloyl-2-thioethyl phosphonoamidate derivative. Therapeuticagents that can be derivatized to phosphoramidate or phosphonoamidatecompound form include any anti-viral agent that includes, or has beenderivatized to include a reactive group for attachment of thephosphoramidate or phosphonoamidate moiety, including but not limited tonucleosides and nucleoside analogues including acyclic nucleosides. Anyof the phosphoramidate or phosphonoamidate compounds disclosed hereincan be provided in the appropriate pharmaceutical composition and beadministered by a suitable route of administration.

The methods provided herein encompass administering pharmaceuticalcompositions containing at least one compound as described herein,including a compound of general Formula I, IIa or IIb, if appropriate inthe salt form, either used alone or in the form of a combination withone or more compatible and pharmaceutically acceptable carriers, such asdiluents or adjuvants, or with another anti-HCV or anti-HBV agent.

In certain embodiments, the second agent can be formulated or packagedwith the compound provided herein. Of course, the second agent will onlybe formulated with the compound provided herein when, according to thejudgment of those of skill in the art, such co-formulation should notinterfere with the activity of either agent or the method ofadministration. In certain embodiments, the compound provided herein andthe second agent are formulated separately. They can be packagedtogether, or packaged separately, for the convenience of thepractitioner of skill in the art.

In clinical practice the active agents provided herein may beadministered by any conventional route, in particular orally,parenterally, rectally or by inhalation (e.g. in the form of aerosols).In certain embodiments, the compound provided herein is administeredorally.

Use may be made, as solid compositions for oral administration, oftablets, pills, hard gelatin capsules, powders or granules. In thesecompositions, the active product is mixed with one or more inertdiluents or adjuvants, such as sucrose, lactose or starch.

These compositions can comprise substances other than diluents, forexample a lubricant, such as magnesium stearate, or a coating intendedfor controlled release.

Use may be made, as liquid compositions for oral administration, ofsolutions which are pharmaceutically acceptable, suspensions, emulsions,syrups and elixirs containing inert diluents, such as water or liquidparaffin. These compositions can also comprise substances other thandiluents, for example wetting, sweetening or flavoring products.

The compositions for parenteral administration can be emulsions orsterile solutions. Use may be made, as solvent or vehicle, of propyleneglycol, a polyethylene glycol, vegetable oils, in particular olive oil,or injectable organic esters, for example ethyl oleate. Thesecompositions can also contain adjuvants, in particular wetting,isotonizing, emulsifying, dispersing and stabilizing agents.Sterilization can be carried out in several ways, for example using abacteriological filter, by radiation or by heating. They can also beprepared in the form of sterile solid compositions which can bedissolved at the time of use in sterile water or any other injectablesterile medium.

The compositions for rectal administration are suppositories or rectalcapsules which contain, in addition to the active principle, excipientssuch as cocoa butter, semi-synthetic glycerides or polyethylene glycols.

The compositions can also be aerosols. For use in the form of liquidaerosols, the compositions can be stable sterile solutions or solidcompositions dissolved at the time of use in apyrogenic sterile water,in saline or any other pharmaceutically acceptable vehicle. For use inthe form of dry aerosols intended to be directly inhaled, the activeprinciple is finely divided and combined with a water-soluble soliddiluent or vehicle, for example dextran, mannitol or lactose.

In one embodiment, a composition provided herein is a pharmaceuticalcomposition or a single unit dosage form. Pharmaceutical compositionsand single unit dosage forms provided herein comprise a prophylacticallyor therapeutically effective amount of one or more prophylactic ortherapeutic agents (e.g., a compound provided herein, or otherprophylactic or therapeutic agent), and a typically one or morepharmaceutically acceptable carriers or excipients. In a specificembodiment and in this context, the term “pharmaceutically acceptable”means approved by a regulatory agency of the Federal or a stategovernment or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” includes a diluent, adjuvant (e.g., Freund'sadjuvant (complete and incomplete)), excipient, or vehicle with whichthe therapeutic is administered. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Water can be used as a carrierwhen the pharmaceutical composition is administered intravenously.Saline solutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well-known to those skilled inthe art of pharmacy, and non limiting examples of suitable excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a subjectand the specific active ingredients in the dosage form. The compositionor single unit dosage form, if desired, can also contain minor amountsof wetting or emulsifying agents, or pH buffering agents.

Lactose free compositions provided herein can comprise excipients thatare well known in the art and are listed, for example, in the U.S.Pharmocopia (USP)SP (XXI)/NF (XVI). In general, lactose freecompositions comprise an active ingredient, a binder/filler, and alubricant in pharmaceutically compatible and pharmaceutically acceptableamounts. Exemplary lactose free dosage forms comprise an activeingredient, microcrystalline cellulose, pre gelatinized starch, andmagnesium stearate.

Further encompassed herein are anhydrous pharmaceutical compositions anddosage forms comprising active ingredients, since water can facilitatethe degradation of some compounds. For example, the addition of water(e.g., 5%) is widely accepted in the pharmaceutical arts as a means ofsimulating long term storage in order to determine characteristics suchas shelf life or the stability of formulations over time. See, e.g.,Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed.,Marcel Dekker, NY, N.Y., 1995, pp. 379 80. In effect, water and heataccelerate the decomposition of some compounds. Thus, the effect ofwater on a formulation can be of great significance since moistureand/or humidity are commonly encountered during manufacture, handling,packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms provided hereincan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine can be anhydrousif substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions can be packaged using materials known to prevent exposureto water such that they can be included in suitable formulary kits.Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs, and strip packs.

Further provided are pharmaceutical compositions and dosage forms thatcomprise one or more compounds that reduce the rate by which an activeingredient will decompose. Such compounds, which are referred to hereinas “stabilizers,” include, but are not limited to, antioxidants such asascorbic acid, pH buffers, or salt buffers.

The pharmaceutical compositions and single unit dosage forms can takethe form of solutions, suspensions, emulsion, tablets, pills, capsules,powders, sustained-release formulations and the like. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Such compositions and dosage forms willcontain a prophylactically or therapeutically effective amount of aprophylactic or therapeutic agent, in certain embodiments, in purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the subject. The formulation shouldsuit the mode of administration. In a certain embodiment, thepharmaceutical compositions or single unit dosage forms are sterile andin suitable form for administration to a subject, for example, an animalsubject, such as a mammalian subject, for example, a human subject.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude, but are not limited to, parenteral, e.g., intravenous,intradermal, subcutaneous, intramuscular, subcutaneous, oral, buccal,sublingual, inhalation, intranasal, transdermal, topical, transmucosal,intra-tumoral, intra-synovial and rectal administration. In a specificembodiment, the composition is formulated in accordance with routineprocedures as a pharmaceutical composition adapted for intravenous,subcutaneous, intramuscular, oral, intranasal or topical administrationto human beings. In an embodiment, a pharmaceutical composition isformulated in accordance with routine procedures for subcutaneousadministration to human beings. Typically, compositions for intravenousadministration are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition may also include a solubilizing agent and alocal anesthetic such as lignocamne to ease pain at the site of theinjection.

Examples of dosage forms include, but are not limited to: tablets;caplets; capsules, such as soft elastic gelatin capsules; cachets;troches; lozenges; dispersions; suppositories; ointments; cataplasms(poultices); pastes; powders; dressings; creams; plasters; solutions;patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosageforms suitable for oral or mucosal administration to a subject,including suspensions (e.g., aqueous or non aqueous liquid suspensions,oil in water emulsions, or a water in oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a subject; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a subject.

The composition, shape, and type of dosage forms provided herein willtypically vary depending on their use. For example, a dosage form usedin the initial treatment of viral infection may contain larger amountsof one or more of the active ingredients it comprises than a dosage formused in the maintenance treatment of the same infection. Similarly, aparenteral dosage form may contain smaller amounts of one or more of theactive ingredients it comprises than an oral dosage form used to treatthe same disease or disorder. These and other ways in which specificdosage forms encompassed herein will vary from one another will bereadily apparent to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa. (2000).

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

Typical dosage forms comprise a compound provided herein, or apharmaceutically acceptable salt, solvate or hydrate thereof lie withinthe range of from about 0.1 mg to about 1000 mg per day, given as asingle once-a-day dose in the morning or as divided doses throughout theday taken with food. Particular dosage forms can have about 0.1, 0.2,0.3, 0.4, 0.5, 1.0, 2.0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 100,200, 250, 500 or 1000 mg of the active compound.

Oral Dosage Forms

Pharmaceutical compositions that are suitable for oral administrationcan be presented as discrete dosage forms, such as, but are not limitedto, tablets (e.g., chewable tablets), caplets, capsules, and liquids(e.g., flavored syrups). Such dosage forms contain predetermined amountsof active ingredients, and may be prepared by methods of pharmacy wellknown to those skilled in the art. See generally, Remington'sPharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa. (2000).

In certain embodiments, the oral dosage forms are solid and preparedunder anhydrous conditions with anhydrous ingredients, as described indetail in the sections above. However, the scope of the compositionsprovided herein extends beyond anhydrous, solid oral dosage forms. Assuch, further forms are described herein.

Typical oral dosage forms are prepared by combining the activeingredient(s) in an intimate admixture with at least one excipientaccording to conventional pharmaceutical compounding techniques.Excipients can take a wide variety of forms depending on the form ofpreparation desired for administration. For example, excipients suitablefor use in oral liquid or aerosol dosage forms include, but are notlimited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms, in which case solidexcipients are employed. If desired, tablets can be coated by standardaqueous or nonaqueous techniques. Such dosage forms can be prepared byany of the methods of pharmacy. In general, pharmaceutical compositionsand dosage forms are prepared by uniformly and intimately admixing theactive ingredients with liquid carriers, finely divided solid carriers,or both, and then shaping the product into the desired presentation ifnecessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms include,but are not limited to, binders, fillers, disintegrants, and lubricants.Binders suitable for use in pharmaceutical compositions and dosage formsinclude, but are not limited to, corn starch, potato starch, or otherstarches, gelatin, natural and synthetic gums such as acacia, sodiumalginate, alginic acid, other alginates, powdered tragacanth, guar gum,cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate,carboxymethyl cellulose calcium, sodium carboxymethyl cellulose),polyvinyl pyrrolidone, methyl cellulose, pre gelatinized starch,hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910),microcrystalline cellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions is typically presentin from about 50 to about 99 weight percent of the pharmaceuticalcomposition or dosage form.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL PH 101, AVICEL PH 103 AVICEL RC581, AVICEL PH 105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. Anspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC 581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL PH 103™ and Starch 1500LM.

Disintegrants are used in the compositions to provide tablets thatdisintegrate when exposed to an aqueous environment. Tablets thatcontain too much disintegrant may disintegrate in storage, while thosethat contain too little may not disintegrate at a desired rate or underthe desired conditions. Thus, a sufficient amount of disintegrant thatis neither too much nor too little to detrimentally alter the release ofthe active ingredients should be used to form solid oral dosage forms.The amount of disintegrant used varies based upon the type offormulation, and is readily discernible to those of ordinary skill inthe art. Typical pharmaceutical compositions comprise from about 0.5 toabout 15 weight percent of disintegrant, specifically from about 1 toabout 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms include, but are not limited to, agar agar, alginic acid, calciumcarbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, polacrilin potassium, sodium starch glycolate, potato ortapioca starch, pre gelatinized starch, other starches, clays, otheralgins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms include, but are not limited to, calcium stearate, magnesiumstearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol,polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate,talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zincstearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof.Additional lubricants include, for example, a syloid silica gel (AEROSIL200, manufactured by W.R. Grace Co. of Baltimore, Md.), a coagulatedaerosol of synthetic silica (marketed by Degussa Co. of Plano, Tex.),CAB O SIL (a pyrogenic silicon dioxide product sold by Cabot Co. ofBoston, Mass.), and mixtures thereof. If used at all, lubricants aretypically used in an amount of less than about 1 weight percent of thepharmaceutical compositions or dosage forms into which they areincorporated.

Delayed Release Dosage Forms

Active ingredients such as the compounds provided herein can beadministered by controlled release means or by delivery devices that arewell known to those of ordinary skill in the art. Examples include, butare not limited to, those described in U.S. Pat. Nos. 3,845,770;3,916,899; 3,536,809; 3,598,123; and 4,008,719; 5,674,533; 5,059,595;5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480;5,733,566; 5,739,108; 5,891,474; 5,922,356; 5,972,891; 5,980,945;5,993,855; 6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363;6,264,970; 6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358;6,699,500 each of which is incorporated herein by reference. Such dosageforms can be used to provide slow or controlled release of one or moreactive ingredients using, for example, hydropropylmethyl cellulose,other polymer matrices, gels, permeable membranes, osmotic systems,multilayer coatings, microparticles, liposomes, microspheres, or acombination thereof to provide the desired release profile in varyingproportions. Suitable controlled release formulations known to those ofordinary skill in the art, including those described herein, can bereadily selected for use with the active ingredients provided herein.Thus encompasseed herein are single unit dosage forms suitable for oraladministration such as, but not limited to, tablets, capsules, gelcaps,and caplets that are adapted for controlled release.

All controlled release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non controlledcounterparts. Ideally, the use of an optimally designed controlledrelease preparation in medical treatment is characterized by a minimumof drug substance being employed to cure or control the condition in aminimum amount of time. Advantages of controlled release formulationsinclude extended activity of the drug, reduced dosage frequency, andincreased subject compliance. In addition, controlled releaseformulations can be used to affect the time of onset of action or othercharacteristics, such as blood levels of the drug, and can thus affectthe occurrence of side (e.g., adverse) effects.

Most controlled release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds. In certain embodiments, the drug may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (see, Sefton, CRC Crit. Ref. Biomed. Eng.14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N.Engl. J. Med. 321:574 (1989)). In another embodiment, polymericmaterials can be used. In yet another embodiment, a controlled releasesystem can be placed in a subject at an appropriate site determined by apractitioner of skill, i.e., thus requiring only a fraction of thesystemic dose (see, e.g., Goodson, Medical Applications of ControlledRelease, vol. 2, pp. 115-138 (1984)). Other controlled release systemsare discussed in the review by Langer (Science 249:1527-1533 (1990)).The active ingredient can be dispersed in a solid inner matrix, e.g.,polymethylmethacrylate, polybutylmethacrylate, plasticized orunplasticized polyvinylchloride, plasticized nylon, plasticizedpolyethyleneterephthalate, natural rubber, polyisoprene,polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetatecopolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonatecopolymers, hydrophilic polymers such as hydrogels of esters of acrylicand methacrylic acid, collagen, cross-linked polyvinylalcohol andcross-linked partially hydrolyzed polyvinyl acetate, that is surroundedby an outer polymeric membrane, e.g., polyethylene, polypropylene,ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,ethylene/vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride,vinylchloride copolymers with vinyl acetate, vinylidene chloride,ethylene and propylene, ionomer polyethylene terephthalate, butyl rubberepichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer, that is insoluble in body fluids.The active ingredient then diffuses through the outer polymeric membranein a release rate controlling step. The percentage of active ingredientin such parenteral compositions is highly dependent on the specificnature thereof, as well as the needs of the subject.

Parenteral Dosage Forms

In one embodiment, provided are parenteral dosage forms. Parenteraldosage forms can be administered to subjects by various routesincluding, but not limited to, subcutaneous, intravenous (includingbolus injection), intramuscular, and intraarterial. Because theiradministration typically bypasses subjects' natural defenses againstcontaminants, parenteral dosage forms are typically, sterile or capableof being sterilized prior to administration to a subject. Examples ofparenteral dosage forms include, but are not limited to, solutions readyfor injection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage formsare well known to those skilled in the art. Examples include, but arenot limited to: Water for Injection USP; aqueous vehicles such as, butnot limited to, Sodium Chloride Injection, Ringer's Injection, DextroseInjection, Dextrose and Sodium Chloride Injection, and Lactated Ringer'sInjection; water miscible vehicles such as, but not limited to, ethylalcohol, polyethylene glycol, and polypropylene glycol; and non aqueousvehicles such as, but not limited to, corn oil, cottonseed oil, peanutoil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms.

Transdermal, Topical & Mucosal Dosage Forms

Also provided are transdermal, topical, and mucosal dosage forms.Transdermal, topical, and mucosal dosage forms include, but are notlimited to, ophthalmic solutions, sprays, aerosols, creams, lotions,ointments, gels, solutions, emulsions, suspensions, or other forms knownto one of skill in the art. See, e.g., Remington's PharmaceuticalSciences, 16^(th), 18th and 20^(th) eds., Mack Publishing, Easton Pa.(1980, 1990 & 2000); and Introduction to Pharmaceutical Dosage Forms,4th ed., Lea & Febiger, Philadelphia (1985). Dosage forms suitable fortreating mucosal tissues within the oral cavity can be formulated asmouthwashes or as oral gels. Further, transdermal dosage forms include“reservoir type” or “matrix type” patches, which can be applied to theskin and worn for a specific period of time to permit the penetration ofa desired amount of active ingredients.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide transdermal, topical, and mucosal dosageforms encompassed herein are well known to those skilled in thepharmaceutical arts, and depend on the particular tissue to which agiven pharmaceutical composition or dosage form will be applied. Withthat fact in mind, typical excipients include, but are not limited to,water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3diol, isopropyl myristate, isopropyl palmitate, mineral oil, andmixtures thereof to form lotions, tinctures, creams, emulsions, gels orointments, which are non toxic and pharmaceutically acceptable.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art. See, e.g., Remington'sPharmaceutical Sciences, 16^(th), 18th and 20^(th) eds., MackPublishing, Easton Pa. (1980, 1990 & 2000).

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith active ingredients provided. For example, penetration enhancers canbe used to assist in delivering the active ingredients to the tissue.Suitable penetration enhancers include, but are not limited to: acetone;various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkylsulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethylformamide; polyethylene glycol; pyrrolidones such aspolyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; andvarious water soluble or insoluble sugar esters such as Tween 80(polysorbate 80) and Span 60 (sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery enhancing orpenetration enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

Dosage and Unit Dosage Forms

In human therapeutics, the doctor will determine the posology which heconsiders most appropriate according to a preventive or curativetreatment and according to the age, weight, stage of the infection andother factors specific to the subject to be treated. In certainembodiments, doses are from about 1 to about 1000 mg per day for anadult, or from about 5 to about 250 mg per day or from about 10 to 50 mgper day for an adult. In certain embodiments, doses are from about 5 toabout 400 mg per day or 25 to 200 mg per day per adult. In certainembodiments, dose rates of from about 50 to about 500 mg per day arealso contemplated.

In further aspects, provided are methods of treating or preventing anHCV and/or HBV infection in a subject by administering, to a subject inneed thereof, an effective amount of a compound provided herein, or apharmaceutically acceptable salt thereof. The amount of the compound orcomposition which will be effective in the prevention or treatment of adisorder or one or more symptoms thereof will vary with the nature andseverity of the disease or condition, and the route by which the activeingredient is administered. The frequency and dosage will also varyaccording to factors specific for each subject depending on the specifictherapy (e.g., therapeutic or prophylactic agents) administered, theseverity of the disorder, disease, or condition, the route ofadministration, as well as age, body, weight, response, and the pastmedical history of the subject. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

In certain embodiments, exemplary doses of a composition includemilligram or microgram amounts of the active compound per kilogram ofsubject or sample weight (e.g., about 10 micrograms per kilogram toabout 50 milligrams per kilogram, about 100 micrograms per kilogram toabout 25 milligrams per kilogram, or about 100 microgram per kilogram toabout 10 milligrams per kilogram). For compositions provided herein, incertain embodiments, the dosage administered to a subject is 0.140 mg/kgto 3 mg/kg of the subject's body weight, based on weight of the activecompound. In certain embodiments, the dosage administered to a subjectis between 0.20 mg/kg and 2.00 mg/kg, or between 0.30 mg/kg and 1.50mg/kg of the subject's body weight.

In certain embodiments, the recommended daily dose range of acomposition provided herein for the conditions described herein liewithin the range of from about 0.1 mg to about 1000 mg per day, given asa single once-a-day dose or as divided doses throughout a day. In oneembodiment, the daily dose is administered twice daily in equallydivided doses. In certain embodiments, a daily dose range should be fromabout 10 mg to about 200 mg per day, in other embodiments, between about10 mg and about 150 mg per day, in further embodiments, between about 25and about 100 mg per day. It may be necessary to use dosages of theactive ingredient outside the ranges disclosed herein in some cases, aswill be apparent to those of ordinary skill in the art. Furthermore, itis noted that the clinician or treating physician will know how and whento interrupt, adjust, or terminate therapy in conjunction with subjectresponse.

Different therapeutically effective amounts may be applicable fordifferent diseases and conditions, as will be readily known by those ofordinary skill in the art. Similarly, amounts sufficient to prevent,manage, treat or ameliorate such disorders, but insufficient to cause,or sufficient to reduce, adverse effects associated with the compositionprovided herein are also encompassed by the above described dosageamounts and dose frequency schedules. Further, when a subject isadministered multiple dosages of a composition provided herein, not allof the dosages need be the same. For example, the dosage administered tothe subject may be increased to improve the prophylactic or therapeuticeffect of the composition or it may be decreased to reduce one or moreside effects that a particular subject is experiencing.

In certain embodiment, the dosage of the composition provided herein,based on weight of the active compound, administered to prevent, treat,manage, or ameliorate a disorder, or one or more symptoms thereof in asubject is 0.1 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6mg/kg, 10 mg/kg, or 15 mg/kg or more of a subject's body weight. Inanother embodiment, the dosage of the composition or a compositionprovided herein administered to prevent, treat, manage, or ameliorate adisorder, or one or more symptoms thereof in a subject is a unit dose of0.1 mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg,0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg,1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

In certain embodiments, treatment or prevention can be initiated withone or more loading doses of a compound or composition provided hereinfollowed by one or more maintenance doses. In such embodiments, theloading dose can be, for instance, about 60 to about 400 mg per day, orabout 100 to about 200 mg per day for one day to five weeks. The loadingdose can be followed by one or more maintenance doses. In certainembodiments, each maintenance does is, independently, about from about10 mg to about 200 mg per day, between about 25 mg and about 150 mg perday, or between about 25 and about 80 mg per day. Maintenance doses canbe administered daily and can be administered as single doses, or asdivided doses.

In certain embodiments, a dose of a compound or composition providedherein can be administered to achieve a steady-state concentration ofthe active ingredient in blood or serum of the subject. The steady-stateconcentration can be determined by measurement according to techniquesavailable to those of skill or can be based on the physicalcharacteristics of the subject such as height, weight and age. Incertain embodiments, a sufficient amount of a compound or compositionprovided herein is administered to achieve a steady-state concentrationin blood or serum of the subject of from about 300 to about 4000 ng/mL,from about 400 to about 1600 ng/mL, or from about 600 to about 1200ng/mL. In some embodiments, loading doses can be administered to achievesteady-state blood or serum concentrations of about 1200 to about 8000ng/mL, or about 2000 to about 4000 ng/mL for one to five days. Incertain embodiments, maintenance doses can be administered to achieve asteady-state concentration in blood or serum of the subject of fromabout 300 to about 4000 ng/mL, from about 400 to about 1600 ng/mL, orfrom about 600 to about 1200 ng/mL.

In certain embodiments, administration of the same composition may berepeated and the administrations may be separated by at least 1 day, 2days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75days, 3 months, or 6 months. In other embodiments, administration of thesame prophylactic or therapeutic agent may be repeated and theadministration may be separated by at least at least 1 day, 2 days, 3days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3months, or 6 months.

In certain aspects, provided herein are unit dosages comprising acompound, or a pharmaceutically acceptable salt thereof, in a formsuitable for administration. Such forms are described in detail above.In certain embodiments, the unit dosage comprises 1 to 1000 mg, 5 to 250mg or 10 to 50 mg active ingredient. In particular embodiments, the unitdosages comprise about 1, 5, 10, 25, 50, 100, 125, 250, 500 or 1000 mgactive ingredient. Such unit dosages can be prepared according totechniques familiar to those of skill in the art.

The dosages of the second agents are to be used in the combinationtherapies provided herein. In certain embodiments, dosages lower thanthose which have been or are currently being used to prevent or treatHCV and/or HBV infection are used in the combination therapies providedherein. The recommended dosages of second agents can be obtained fromthe knowledge of those of skill. For those second agents that areapproved for clinical use, recommended dosages are described in, forexample, Hardman et al., eds., 1996, Goodman & Gilman's ThePharmacological Basis Of Basis Of Therapeutics 9^(th) Ed, McGraw-Hill,New York; Physician's Desk Reference (PDR) 57^(th) Ed., 2003, MedicalEconomics Co., Inc., Montvale, N.J., which are incorporated herein byreference in its entirety.

In various embodiments, the therapies (e.g., a compound provided hereinand the second agent) are administered less than 5 minutes apart, lessthan 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1to about 2 hours apart, at about 2 hours to about 3 hours apart, atabout 3 hours to about 4 hours apart, at about 4 hours to about 5 hoursapart, at about 5 hours to about 6 hours apart, at about 6 hours toabout 7 hours apart, at about 7 hours to about 8 hours apart, at about 8hours to about 9 hours apart, at about 9 hours to about 10 hours apart,at about 10 hours to about 11 hours apart, at about 11 hours to about 12hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hoursapart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hoursto 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hoursapart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96hours to 120 hours part. In various embodiments, the therapies areadministered no more than 24 hours apart or no more than 48 hours apart.In certain embodiments, two or more therapies are administered withinthe same patient visit. In other embodiments, the compound providedherein and the second agent are administered concurrently.

In other embodiments, the compound provided herein and the second agentare administered at about 2 to 4 days apart, at about 4 to 6 days apart,at about 1 week part, at about 1 to 2 weeks apart, or more than 2 weeksapart.

In certain embodiments, administration of the same agent may be repeatedand the administrations may be separated by at least 1 day, 2 days, 3days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3months, or 6 months. In other embodiments, administration of the sameagent may be repeated and the administration may be separated by atleast at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days,45 days, 2 months, 75 days, 3 months, or 6 months.

In certain embodiments, a compound provided herein and a second agentare administered to a patient, for example, a mammal, such as a human,in a sequence and within a time interval such that the compound providedherein can act together with the other agent to provide an increasedbenefit than if they were administered otherwise. For example, thesecond active agent can be administered at the same time or sequentiallyin any order at different points in time; however, if not administeredat the same time, they should be administered sufficiently close in timeso as to provide the desired therapeutic or prophylactic effect. In oneembodiment, the compound provided herein and the second active agentexert their effect at times which overlap. Each second active agent canbe administered separately, in any appropriate form and by any suitableroute. In other embodiments, the compound provided herein isadministered before, concurrently or after administration of the secondactive agent.

In certain embodiments, the compound provided herein and the secondagent are cyclically administered to a patient. Cycling therapy involvesthe administration of a first agent (e.g., a first prophylactic ortherapeutic agents) for a period of time, followed by the administrationof a second agent and/or third agent (e.g., a second and/or thirdprophylactic or therapeutic agents) for a period of time and repeatingthis sequential administration. Cycling therapy can reduce thedevelopment of resistance to one or more of the therapies, avoid orreduce the side effects of one of the therapies, and/or improve theefficacy of the treatment.

In certain embodiments, the compound provided herein and the secondactive agent are administered in a cycle of less than about 3 weeks,about once every two weeks, about once every 10 days or about once everyweek. One cycle can comprise the administration of a compound providedherein and the second agent by infusion over about 90 minutes everycycle, about 1 hour every cycle, about 45 minutes every cycle. Eachcycle can comprise at least 1 week of rest, at least 2 weeks of rest, atleast 3 weeks of rest. The number of cycles administered is from about 1to about 12 cycles, more typically from about 2 to about 10 cycles, andmore typically from about 2 to about 8 cycles.

In other embodiments, courses of treatment are administered concurrentlyto a patient, i.e., individual doses of the second agent areadministered separately yet within a time interval such that thecompound provided herein can work together with the second active agent.For example, one component can be administered once per week incombination with the other components that can be administered onceevery two weeks or once every three weeks. In other words, the dosingregimens are carried out concurrently even if the therapeutics are notadministered simultaneously or during the same day.

The second agent can act additively or synergistically with the compoundprovided herein. In one embodiment, the compound provided herein isadministered concurrently with one or more second agents in the samepharmaceutical composition. In another embodiment, a compound providedherein is administered concurrently with one or more second agents inseparate pharmaceutical compositions. In still another embodiment, acompound provided herein is administered prior to or subsequent toadministration of a second agent. Also contemplated are administrationof a compound provided herein and a second agent by the same ordifferent routes of administration, e.g., oral and parenteral. Incertain embodiments, when the compound provided herein is administeredconcurrently with a second agent that potentially produces adverse sideeffects including, but not limited to, toxicity, the second active agentcan advantageously be administered at a dose that falls below thethreshold that the adverse side effect is elicited.

Kits

Also provided are kits for use in methods of treatment of a liverdisorder such as HCV and/or HBV infections. The kits can include acompound or composition provided herein, a second agent or composition,and instructions providing information to a health care providerregarding usage for treating the disorder. Instructions may be providedin printed form or in the form of an electronic medium such as a floppydisc, CD, or DVD, or in the form of a website address where suchinstructions may be obtained. A unit dose of a compound or compositionprovided herein, or a second agent or composition, can include a dosagesuch that when administered to a subject, a therapeutically orprophylactically effective plasma level of the compound or compositioncan be maintained in the subject for at least 1 days. In someembodiments, a compound or composition can be included as a sterileaqueous pharmaceutical composition or dry powder (e.g., lyophilized)composition.

In some embodiments, suitable packaging is provided. As used herein,“packaging” includes a solid matrix or material customarily used in asystem and capable of holding within fixed limits a compound providedherein and/or a second agent suitable for administration to a subject.Such materials include glass and plastic (e.g., polyethylene,polypropylene, and polycarbonate) bottles, vials, paper, plastic, andplastic-foil laminated envelopes and the like. If e-beam sterilizationtechniques are employed, the packaging should have sufficiently lowdensity to permit sterilization of the contents.

The following Examples illustrate the synthesis of representativecompounds provided herein. These examples are not intended, nor are theyto be construed, as limiting the scope of the claimed subject matter. Itwill be clear that the scope of claimed subject matter may be practicedotherwise than as particularly described herein. Numerous modificationsand variations of the subject matter are possible in view of theteachings herein and, therefore, are within the scope the claimedsubject matter.

EXAMPLES Example 1 Preparation of A550 (NM204), the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of L-2′,3′-dideoxyadenosine L-ddA

Synthetic Scheme:

Synthesis of Carboxylic Acid 2:

2,2-Dimethyl-3-hydroxypropanoic acid methyl ester (965 μL, 7.57 mmol)was added dropwise to a stirring solution of 4,4′-dimethoxytritylchloride (2.82 g, 8.33 mmol) in anhydrous pyridine (7.6 mL) at roomtemperature. The reaction mixture turned to a red solution quickly, thento an orange suspension (ca. 30 min), and this was left stirringovernight. The mixture was poured carefully over saturated aqueousNaHCO₃ solution (30 mL) and the product was extracted with Et₂O (3×20mL). The combined organic extracts were washed with brine (20 mL), dried(Na₂SO₄) and the volatiles were removed under reduced pressure. Theresulting oil was co-evaporated with toluene and the residue was quicklypurified by flash column chromatography (SiO₂, Ø=4 cm, H=20 cm) elutingwith 5→10→20→30% Et₂O in petroleum ether (40-60). Evaporation of thefractions (R_(f)=0.25, 30% Et₂O in petroleum ether (40-60)) affordedether 1 as a yellow oil (3.11 g, 95%). This compound (3.00 g, 6.91 mmol)was dissolved in THF (35 mL) and an aqueous solution of NaOH (10%, 3.5 gin 35 mL H₂O) was then added at room temperature. The solution turnedinstantly dark orange and this was stirred for 2 days. The medium wasthen carefully neutralized by dropwise addition of HCl (1M). The productwas extracted with Et₂O (4×50 mL) and the combined organic extracts werewashed with brine (50 mL), dried (Na₂SO₄) and the volatiles were removedunder reduced pressure. The crude yellow oil was quickly purified byflash column chromatography (SiO₂, Ø=2 cm, H=10 cm) eluting with 50%Et₂O in petroleum ether (40-60). Evaporation of the fractions affordedcarboxylic acid 2 as a white foam (2.23 g, 77%). R_(f)=0.50 (50% Et₂O inpetroleum ether (40-60)); ¹H-NMR (300 MHz, CDCl₃) 1.10 (s, 6H, 2×CH₃),3.06 (s, 2H, CH₂O), 3.65 (s, 6H, 2×OCH₃), 6.62-6.79 (m, 4H, PhCH),7.02-7.46 (stack, 8H, PhCH); ¹³C-NMR (75 MHz, CDCl₃) 22.6 (2×CH₃), 43.5(C(CH₃)₂), 55.1 (2×OCH₃), 85.9 (CPh₃), [125.3, 126.7, 127.7, 128.2,129.1, 130.0, 136.0, 144.9, 158.4 (Ph), some overlap], 182.2 (C═O).

Synthesis of Thioester 3:

1,1′-carbonyldiimidazole (830 mg, 5.12 mmol) was added to a stirringsolution of carboxylic acid 2 in anhydrous PhMe/DMF (2/1, v/v, 2.7 mL)at room temperature and the reaction mixture turned turbid instantly.After 30 min, the medium was diluted by adding anhydrous PhMe/DMF (93/7,v/v, 17 mL) and cooled to −10° C. 2-Mercaptoethanol (359 μL, 5.12 mmol)was then added dropwise and the solution was stirred for 1 h at thistemperature. The reaction mixture was diluted with H₂O (60 mL) and theproduct was extracted with Et₂O (3×15 mL). The combined organic extractswere washed with brine (15 mL), dried (Na₂SO₄) and the volatiles wereremoved under reduced pressure (bath temperature not exceeding 20° C.).The residue was purified by flash column chromatography (SiO₂, Ø=4 cm,H=15 cm, 1% Et₃N) eluting with 60→70% Et₂O in petroleum ether (40-60).Evaporation of the fractions afforded thioester 3 as a white syrup (1.74g, 92%) that solidified upon storage at 4° C. R_(f)=0.35 (70% Et₂O inpetroleum ether (40-60)); ¹H-NMR (300 MHz, CDCl₃) 1.16 (s, 6H, 2×CH₃),3.02 (t, J 6.0, 2H, CH₂S), 3.09 (s, 2H, CH₂O), 3.66 (t, J 6.0, 2H,CH₂OH), 3.72 (s, 6H, 2×OCH₃), 6.74-6.78 (m, 4H, PhCH), 7.09-7.36 (stack,8H, PhCH); ¹³C-NMR (75 MHz, CDCl₃) 22.9 (CH₃, 2×CH₃), 31.7 (CH₂, CH₂S),51.0 (quat. C, C(CH₃)₂), 55.2 (CH₃, 2×OCH₃), 61.9 (CH₂, CH₂OH), 70.0(CH₂, CH₂O), 85.8 (quat. C., CPh₃), [113.0 (CH, Ph), 126.7 (CH, Ph),127.7 (CH, Ph), 128.2 (CH, Ph), 130.1 (CH, Ph), some overlap], [135.9(quat. C, Ph), 144.8 (quat. C, Ph), 158.4 (quat. C, Ph), some overlap],205.0 (quat. C, C═O).

Synthesis of H-phosphonate Monoester 4:

β-L-ddA (1.00 g, 4.25 mmol) was co-evaporated with anhydrous pyridine(3×10 mL) and then dissolved in anhydrous pyridine/DMF (1/1, v/v, 21mL). Diphenyl phosphite (5.76 mL, 29.8 mmol) was then added dropwise tothis solution at room temperature. The reaction mixture was stirred for20 min upon which a mixture of Et₃N/H₂O (1/1, v/v, 8.5 mL) was addeddropwise, and stirring was pursued for an additional 20 min. Thereaction mixture was concentrated under reduced pressure toapproximately 15-20 mL and this residue was directly purified by flashcolumn chromatography (SiO₂, Ø=4 cm, H=15 cm, 1% Et₃N) eluting slowlywith CH₂Cl₂ (150 mL) then 5% (200 mL)→10% (200 mL)→15% (300 mL) MeOH inCH₂Cl₂. Evaporation of the fractions afforded H-phosphonate monoester 4as a white foam (1.36 g, 80%) that could be kept for several weeks at 4°C. R_(f)=0.10 (Et₃N/MeOH/CH₂Cl₂, Jan. 10, 1989); ¹H-NMR (300 MHz, CDCl₃)1.21 (t, J 7.4, 9H, 3×NCH₂CH₃), 1.92-2.50 (stack, 4H, 2×2′-H, 2×3′-H),3.02 (q, J 7.4, 6H, 3×NCH₂CH₃), [3.96-4.03 and 4.18-4.30 (stacks, 3H,4′-H, 2×5′-H), 6.28 (m, 1′-H), 6.91 (d, J 623, 1H, P—H), 7.05 (br s, 2H,NH₂), 8.21 (s, 1H), 8.54 (br s, 1H, OH), 8.57 (s, 1H).

Synthesis of Phosphoramidate Diester 5

H-Phosphonate monoester 4 (1.03 g, 2.57 mmol) and alcohol 3 (1.66 g,3.45 mmol) were co-evaporated with anhydrous pyridine (3×5 mL) and thendissolved in anhydrous pyridine (5 mL). PyBOP(1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate,1.60 g, 3.08 mmol) was then added in one portion and the reactionmixture was stirred for 15 min at room temperature. The solution waspoured over saturated aqueous NaHCO₃ solution (30 mL) and the productwas extracted with CH₂Cl₂ (4×15 mL). The combined organic extracts werewashed with brine (10 mL), dried (Na₂SO₄) and concentrated under reducedpressure to leave the corresponding H-phosphonate diester as a slightlyyellow oil (1.84 g, assuming 2.41 mmol). This was co-evaporated withanhydrous pyridine (3×5 mL; note: do not evaporate to dryness in orderto help further solubilization), and the residue was dissolved inanhydrous CCl₄ (24 mL). Benzylamine (791 μl, 7.23 mmol) was addeddropwise and the reaction mixture turned cloudy instantly (slight heatdevelopment was observed). The milky solution was stirred for 1 h atroom temperature and poured over saturated aqueous NaHCO₃ solution (30mL) and the product was extracted with CH₂Cl₂ (4×15 mL). The combinedorganic extracts were washed with brine (15 mL), dried (Na₂SO₄) andconcentrated under reduced pressure to afford phosphoramidate diester 5as a yellow oil (2.00 g, assuming 2.31 mmol). This was used in the nextstep without any further purification. R_(f)=0.29 (4% MeOH in CH₂Cl₂);¹H-NMR (300 MHz, CDCl₃) 1.11 (s, 6H, 2×CH₃), 1.91-2.05 (m, 2H),2.31-2.59 (m, 2H), 3.06 (m, 2H, CH₂S), 3.08 (s, 2H, CH₂ODMTr), 3.69 (s,6H, 2×OCH₃), 3.83-4.28 (stacks, 7H, CH₂O, NCH₂Ph, 4′41, 2×5′-H), 5.71(br s, 1H, NH), 6.18 (m, 1H, 1′-H), 6.69-6.80 (m, 4H, PhCH), 7.02-7.31(stack, 13H, PhCH), 7.90 (s, 1H), 8.01 (s, 1H), 8.23 (s, 2H, NH₂);¹³P-NMR (61 MHz, CDCl₃) 8.82, 8.99.

Synthesis of NM204 (A550), the Hydroxy-tBuSATE N-benzylphosphoramidateDerivative of L-ddA:

Crude phosphoramidate diester 5 (2.00 g, assuming 2.31 mmol) wasdissolved in dioxane/AcOH/H₂O (25/17/25, v/v/v, 462 mL) and the solutionwas stirred for 3 d at room temperature. Evaporation of the volatilesunder reduced pressure left a residue that was purified by flash columnchromatography (SiO₂, Ø=3 cm, H=15 cm) eluting with CH₂Cl₂ (100 mL) then2% (100 mL)→4% (100 mL)→6% (100 mL)→8% (150 mL) MeOH in CH₂Cl₂.Evaporation of the fractions left NM 204 as a white foam that wasdissolved in MeCN (5 mL). Upon addition of H₂O (5 mL), the solutionturned turbid and required sonication before lyophilization. Theresulting white powder was dried at room temperature (using P₂O₅ as adesiccant) under vacuum for 1 d. The title compound was obtained as ahighly hygroscopic white powder (1:1 mixture of diastereoisomers asjudged by ³¹P-NMR; 499 mg, 35% over 3 steps). [α]²⁰ _(D)=+4.2° (c 1.0,CHCl₃); R_(f)=0.29 (4% MeOH in CH₂Cl₂); ¹H-NMR (300 MHz, DMSO-d6) 1.10(s, 6H, 2×CH₃), 2.02-2.14 (m, 2H, 2×3′-H), 2.41-2.55 (m, 2H, 2×2′-H),3.01 (t, J 6.4, 2H, CH₂S), 3.43 (d, J 5.0, 2H, CH₂OH), 3.75-4.07 and4.18-4.29 (stacks, 7H, CH₂O, NCH₂Ph, 4′-H, 2×5′-H), 5.02 (t, J 5.0, 1H,OH), 5.62 (m, 1H, NH), 6.25 (t, J 5.1, 1H, 1′-H), 7.16-7.36 (stack, 7H,PhH, NH₂), 8.14 (s, 1H), 8.26 (s, 1H); ¹³C-NMR (75 MHz, DMSO-d6) 21.8(2×CH₃), 25.9 and 26.0 (CH₂, 3′-C), 28.2 and 28.3 (CH₂, CH₂S), 30.9 and31.0 (CH₂, 2′-C), 44.2 (CH₂, NCH₂Ph), 51.7 (quat. C, C(CH₃)₂), 63.7 and63.8 (CH₂, CH₂O), 66.8 (CH₂, m, 5′-C), 68.3 (CH₂, CH₂OH), 78.9 (CH, m,4′-C), 84.2 (CH, 1′-C), 118.9 (quat. C), [126.5 (CH, Ph), 127.2 (CH,Ph), 128.1 (CH, Ph), some overlap], 138.8 and 138.9 (CH), 140.5 and140.6 (quat. C), 148.9 (quat. C), 152.3 (CH), 155.0 (quat. C), 204.0(quat. C, C═O); ¹³P-NMR (61 MHz, DMSO-d6) 9.86, 9.95; m/z (FAB⁻) 563(2), 306 (76), 153 (100); HRMS 565.2034 ([M+H]⁺. C₂₄H₃₄O₆N₆PS requires565.1998); HPLC t_(R)=3.52 min (20% TEAC 20 mM in MeCN); UV (EtOH 95%)λ_(max)=259 (ε_(max) 15900), λ_(min)=224 (ε_(min) 7200).

Example 2 Preparation of B102, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of 2′-C-methylcytidine

Procedure A

Synthesis of H-phosphonate Monoester 5

Synthesis of Carboxylic Acid 3:

To a stirred solution of 2,2-dimethyl-3-hydroxypropanoic acid methylester (1, 15 ml, 117.6 mmol) in a mixture of anhydrous methylenechloride (590 ml) and triethylamine (23 ml), were addedtriphenylmethylene chloride (1.2 eq, 39.3 g) and 4-dimethylaminopyridine(0.1 eq, 1.44 g). The reaction mixture was left refluxing overnight. Themixture was poured carefully over a saturated aqueous NaHCO₃ solutionand the product was extracted with methylene chloride and washed withwater. The combined organic extracts were evaporated under reducedpressure to give crude compound 2 which will be used for the next stepwithout further purification. The resulting oil was dissolved in amixture of dioxan (350 ml) and an aqueous solution of NaOH (30%, 350ml). The heterogene mixture was refluxed for 16 hours. The reactionmixture was allowed to cool down to room temperature, the two phaseswere separated, and the organic phase carefully neutralized by dropwiseaddition of HCl (1M). The product was extracted with methylene chlorideand the organic phases were evaporated under reduced pressure. The crudeorange oil was recrystallized from methylene chloride to affordcarboxylic acid 3 as white crystals (92%). R_(f)=0.50 (70% diethyl etherin petroleum ether); ¹H-NMR (400 MHz, CDCl₃) 1.24 (s, 6H, 2×CH₃), 3.19(s, 2H, CH₂O), 7.2-7.5 (m, 15H, C₆H₅).

Synthesis of H-phosphonate Monoester 5:

1,1′-carbonyldiimidazole (1.3 eq, 1.17 g) was added to a stirringsolution of carboxylic acid 3 (2 g, 5.56 mmol) in an anhydrous mixtureof toluene and dimethylformamide (2/1, v/v, 4.5 ml) at room temperature,and the reaction mixture turned turbid instantly. After 30 min, thereaction mixture was diluted with a mixture of toluene anddimethylformamide (93/7, v/v, 28 ml), cooled to −10° C., and2-mercaptoethanol (1.3 eq, 500 μL) was added. The solution was stirredfor 3 h at this temperature. The volatiles were removed under reducedpressure (bath temperature not exceeding 25° C.). The residue wasdissolved in methylene chloride and washed with water. The organicphases were combined, dried over sodium sulphate (Na₂SO₄), filtered andevaporated to dryness to give compound 4 as a yellow oil. This compoundwill be coevaporated with anhydrous pyridine and used for the next stepwithout further purification. R_(f)=0.71 (70% Et₂O in petroleum ether);¹H-NMR (400 MHz, CDCl₃) 1.20 (s, 6H, 2×CH₃), 3.05 (t, J=6.4 Hz, 2H,CH₂S), 3.15 (s, 2H, CH₂OTr), 3.69 (t, J=6.4 Hz, 2H, CH₂OH), 7.3-7.9 (m,15H, C₆H₅).

Phosphorus acid (10 eq, 4.1 g) was coevaporated two times with anhydrouspyridine, dissolved in that solvent (25 ml) and added to crude 4. Thereaction mixture was stirred at room temperature and a white precipitateappeared after few minutes. The reaction mixture was cooled down to 0°C. and pivaloyl chloride (5.5 eq, 3.4 ml) was added. The reactionmixture was allowed to warm to room temperature and stirred for 3 h. Thereaction was stopped by addition of a solution of triethylammoniumbicarbonate (TEAB 1M, 10 ml) and diluted with ethyl acetate (EtOAc).After extraction with EtOAc and TEAB 0.5M, the organic phases werecombined, dried over sodium sulphate, filtered and evaporated to dryness(bath temperature not exceeding 30° C.). The residue was purified byflash column chromatography eluting with 10% of methanol in methylenechloride+1% triethylamine. Evaporation of the fractions afforded theH-phosphonate monoester 5 as a white syrup (90%). R_(f)=0.25 (70% Et₂Oin petroleum ether); ¹H-NMR (400 MHz, CDCl₃) 1.17 (m, 2×CH₃+excess(CH₃CH₂)₃N), 2.9 (m, excess (CH₃CH₂)₃N), 3.12 (t, J=6.8 Hz, 2H, CH₂S),3.37 (s, 2H, CH₂OTr), 3.90 (m, 2H, CH₂OP), 7.2-7.6 (m, 15H, C₆H₅), 9.9(m, excess (CH₃CH₂)₃NH); ³¹P-NMR (161 MHz, CDCl₃) 3.85 (s).

Synthesis of B102, the Hydroxy-tBuSATE N-benzylphosphoramidateDerivative of 2′-C-methylcytidine:

The following two strategies were used:

Strategy a

Synthesis of the Protected Nucleoside 7

A mixture of 2′C-methylcytidine (NM107) (10 g, 39.0 mmol), triethylorthoformate (8.3 eq, 54 ml) and p-toluenesulfonic acid monohydrate (1eq, 7.4 g) in anhydrous acetone (650 ml), was refluxed overnight undernitrogen atmosphere. The reaction mixture was neutralized with anaqueous ammonia solution (26%) and the precipitate filtered. Thefiltrate was evaporated under reduced pressure and coevaporated withethanol. Purification of the crude mixture on silica gel columnchromatography (eluant: stepwise gradient [0-10%] of methanol inmethylene chloride) led to compound 6 as a pale-yellow solid (86%).R_(f)=0.30 (20% MeOH in methylene chloride), ¹H-NMR (400 MHz, DMSO-d₆)1.06 (s, 3H, CH₃), 1.33 (s, 3H, CH₃), 1.47 (s, 3H, CH₃), 3.6 (m, 2H,H-5′, H-5″), 4.1 (m, 1H, H-4′), 4.41 (d, 1H, H-3′, J=3.2 Hz), 5.16 (t,1H, OH-5′, J=4.0 Hz, D₂O exchangeable), 5.69 (d, 1H, H-5, J=8.0 Hz),6.04 (s, 1H, H-1′), 7.14-7.19 (bd, 2H, NH₂, D₂O exchangeable), 7.74 (d,1H, H-6, J=8.0 Hz); LC/MS Scan ES− 296 (M−H)⁻, Scan ES+ 298 (M+H)⁺,λ_(max)=280.7 nm.

Compound 6 (4.4 g, 14.8 mmol) was dissolved in anhydrous pyridine (74ml) and chlorotrimethylsilane (3 eq, 5.4 ml) was added. The reactionmixture was stirred at room temperature under nitrogen atmosphere for 2h, then 4,4′-dimethoxytrityl chloride (1.5 eq, 7.5 g) and4-dimethylaminopyridine (0.5 eq, 900 mg) were successively added. Thereaction mixture was stirred overnight at room temperature, thenquenched with a saturated aqueous NaHCO₃ solution. The crude product wasextracted with methylene chloride, washed with saturated aq NaHCO₃solution, and water. The combined organic phases were concentrated underreduced pressure, then dissolved in a mixture of dioxan (160 ml) andaqueous ammonia (28%, 29 ml). The solution was heated at 70° C. for 3 hand evaporated to dryness. The crude mixture was purified on silica gelcolumn chromatography (eluant: stepwise gradient of methanol [1-5%] inmethylene chloride) to give protected nucleoside 7 as a yellow solid(81%). R_(f)=0.16 (30% EtOAc in CH₂Cl₂)¹H-NMR (400 MHz, DMSO-d₆) 1.03(s, 3H, CH₃), 1.30 (s, 3H, CH₃), 1.42 (s, 3H, CH₃), 3.5 (m, 2H, H-5′,H-5″), 3.71 (s, 6H, 2×OCH₃), 4.0 (d, 1H, H-4′, J=3.2 Hz), 4.36 (d, 1H,H-3′, J=2.8 Hz), 5.1 (m, 1H, OH-5′, D₂O exchangeable), 5.90 (s, 1H,H-1′), 6.2 (m, 1H, H-5), 6.8-7.2 (m, 13H, DMTr), 7.6 (m, 1H, H-6), 8.32(s, 1H, NH, D₂O exchangeable); LC/MS Scan ES− 598 (M−H)⁻, λ_(max1)=231.7nm, λ_(max2) 283.7 nm.

Synthesis B102 (Compound 10)

Compounds 7 (2.0 g, 3.34 mmol) and 5 (2.2 eq, 4.3 g) were coevaporatedtogether with anhydrous pyridine and dissolved in this solvent (50 ml).Pivaloyl chloride (2.5 eq, 1 ml) was added dropwise and the solutionstirred at room temperature for 2 h 30. The reaction mixture was dilutedwith methylene chloride and neutralized with an aqueous solution ofammonium chloride (NH₄Cl 0.5M). After extraction with methylenechloride/aq NH₄Cl 0.5M, the organic phases were combined, evaporatedunder reduced pressure (bath temperature not exceeding 30° C.) andcoevaporated with toluene. The crude mixture was purified on silica gelcolumn chromatography (eluant: stepwise gradient [0-5%] of methanol inmethylene chloride+2°/∞acetic acid) to afford the desired product 8which was coevaporated with toluene to give a beige foam (94%).R_(f)=0.63 (5% MeOH in CH₂Cl₂); ¹H-NMR (400 MHz, CDCl₃) 1.21 (m, 9H,3CH₃), 1.42 (s, 3H, CH₃), 1.60 (s, 3H, CH₃), 3.13 (m, 2H, CH₂S), 3.17(m, 2H, CH₂OTr), 3.79 (s, 6H, 2×OCH₃), 4.1 (m, 2H, CH₂OP), 4.2-4.3 (m,3H, H-5′, H-5″, H-4′), 5.09 (d, 1H, H-3′, J=7.6 Hz), 5.89 (d, 1H, H-5,J=5.6 Hz), 6.0 (m, 1H, H-1′), 6.8-7.7 (m, 29H, Tr, DMTr, H-6); ¹³P-NMR(161 MHz, CDCl₃) 7.92, 8.55; LC/MS Scan ES+ 1066 (M+H)⁺, Scan ES− 1064(M−H)⁻.

a solution of compound 8 (3.4 g, 3.15 mmol) in anhydrous carbontetrachloride (30 ml) was added dropwise benzylamine (10 eq, 3.4 ml).The reaction mixture was stirred at room temperature for 1 h 30. A whiteprecipitate appeared. The solution was diluted with methylene chlorideand neutralized with an aqueous solution of hydrogen chloride (HCl 1M).After successive extractions with CH₂Cl₂/HCl 1M and CH₂Cl₂/aq NaHCO₃,the organic phases were combined, dried over Na₂SO₄, filtered andevaporated to dryness. The crude mixture was purified on silica gelcolumn chromatography (eluant: stepwise gradient [0-5%] of methanol inmethylene chloride) to give 9 as a yellow foam (87%). R_(f)=0.35 (5%MeOH in methylene chloride); ¹H-NMR (400 MHz, CDCl₃) 1.1-1.2 (m, 9H,3CH₃), 1.40 (s, 3H, CH₃), 1.59 (s, 3H, CH₃), 2.9-3.2 (m, 4H, CH₂OTr,CH₂OS), 3.76 (s, 6H, 2×OCH₃), 3.9-4.4 (m, 8H, CH₂OP, CH₂N, H-3′, H-4′,H-5′, H-5″), 5.0 (m, 1H, H-5), 6.0 (2s, 1H, H-1′), 6.7-7.7 (m, 34H, Tr,DMTr, C₆H₅CH₂, H-6); ¹³P-NMR (161 MHz, CDCl₃) 8.40, 8.8.68; LC/MS ScanES+ 1171 (M+H)⁺.

Finally, compound 9 (2.39 g, 2.04 mmol) was dissolved in a mixture ofmethylene chloride (10 ml) and an aqueous solution of trifluoroaceticacid (90%, 10 ml). The reaction mixture was stirred at 35-40° C. for 2h, then diluted with ethanol (140 ml). The volatiles were evaporatedunder reduced pressure and coevaporated with ethanol. The crude mixturewas purified by silica gel column chromatography (eluant: stepwisegradient of methanol [0-30%] in methylene chloride), followed by apurification on reverse phase chromatography (eluant: stepwise gradientof acetonitrile [0-50%] in water), to give the desired product 10 (B102)(1:1 mixture of diastereoisomers as judged by ³¹P-NMR, 36%) which waslyophilized from a mixture of dioxan/water. Rf=0.34 (15% MeOH inmethylene chloride); ¹H-NMR (400 MHz, DMSO-d6) 0.92 (s, 3H, CH₃), 1.10(s, 6H, 2×CH₃), 3.0 (m, 2H, CH₂S), 3.33 (m, 1H, H-3′), 3.56 (s, 2H,CH₂OH), 3.8-4.0 and 4.05-4.25 (stacks, 7H, CH₂OP, NCH₂Ph, H-4′, H-5′ andH-5″), 4.9 (m, 1H, OH-3′, J=5.4 Hz, D₂O exchangeable), 5.07 (s, 1H,OH-2′, D₂O exchangeable), 5.3 (m, 1H, CH₂OH, D₂O exchangeable), 5.6-5.7(m, 2H, H-5 and NH, D₂O exchangeable), 5.91 (s, 1H, H-1′), 7.3-7.4(stack, 7H, PhH, NH₂, D₂O exchangeable), 7.6 (m, 1H, H-6); ¹³P-NMR (161MHz, DMSO-d6) 9.71, 9.91; HPLC t_(R)=4.67 min (0-100% acetonitrile overa period of 8 min), λ_(max)=274.9; LC/MS Scan ES+ 587 (M+H)⁺.

Strategy b

Synthesis of Protected Nucleoside 11

NM107 (10 g, 38.87 mmol) was dissolved in anhydrous pyridine (194 ml)and chlorotrimethylsilane (4.5 eq, 21.6 ml) was added. The reactionmixture was stirred at room temperature under nitrogen atmosphere for 2h 30, then 4,4′-dimethoxytrityl chloride (1.5 eq, 19.8 g) and4-dimethylaminopyridine (0.5 eq, 2.37 g) were successively added. Thereaction mixture was stirred overnight at room temperature, thenquenched with a saturated aqueous NaHCO₃ solution. The crude product wasextracted with methylene chloride, washed with saturated aq NaHCO₃solution, and water. The combined organic phases were concentrated underreduced pressure, then dissolved in tetrahydrofuran (110 ml). To thatsolution was added tetrabutylammonium fluoride 1M in THF (1 eq, 38.87ml) and the reaction mixture was stirred for 30 min at room temperature.After extraction with EtOAc and water, the organic phases were collectedand evaporated to dryness. The crude mixture was purified on silica gelcolumn chromatography (eluant: stepwise gradient of methanol [0-10%] inmethylene chloride) to give protected nucleoside 11 as a yellow solid(93%). R_(f)=0.32 (10% MeOH in CH₂Cl₂) ¹H-NMR (400 MHz, DMSO-d₆) 0.79(s, 3H, CH₃), 3.56 (m, 2H, H-5′, H-5″), 3.71 (s, 7H, 2×OCH₃, H-4′), 5.0(m, 4H, H-3′, OH-2′, OH-3′, OH-5′, D₂O exchangeable), 5.72 (s, 1H,H-1′), 6.16 (m, 1H, H-5), 6.8-7.2 (m, 13H, DMTr), 7.82 (m, 1H, H-6),8.24 (m, 1H, NH D₂O exchangeable); LC/MS Scan ES− 560 (M+H)⁺, ES-558(M−H)⁻, λ_(max)=284.7 nm.

Synthesis of Protected Phosphoramidate Pronucleotide 13, Precursor 10

Compound 11 (7 g, 12.5 mmol) and 5 (1.5 eq, 11.0 g) were coevaporatedtogether with anhydrous pyridine and dissolved in this solvent (187 ml).Pivaloyl chloride (2.0 eq, 3.08 ml) was added dropwise at −15° C. andthe solution stirred at this temperature for 1 h 30. The reactionmixture was diluted with methylene chloride and neutralized with anaqueous solution of ammonium chloride (NH₄Cl 0.5M). After extractionwith methylene chloride/aq NH₄Cl 0.5M, the organic phases were combined,evaporated under reduced pressure (bath temperature not exceeding 30°C.) and coevaporated with toluene. The crude mixture was purified onsilica gel column chromatography (eluant: stepwise gradient [0-5%] ofmethanol in methylene chloride+0.2% acetic acid) to afford the desiredproduct 12 which was coevaporated with toluene to give a white foam (3.5g, 27%). R_(f)=0.44 (5% MeOH in CH₂Cl₂); ¹H-NMR (400 MHz, DMSO) 0.8 (m,3H, CH₃), 1.14 and 1.06 (2s, 6H, 2CH₃), 3.06 (m, 2H, CH₂S), 3.16 (m, 2H,CH₂OTr), 3.5 (m, 1H, H-3′), 3.70 (m, 6H, 2 OCH₃), 3.90 (m, 1H-4′), 4.03(m, 2H, CH₂OP), 4.24 (m, 2H, H-5′, H-5″), 5.30 and 5.04 (2 ms, 2H, OH-2′and OH-3′, D₂O exchangeable), 5.78 (m, 1H, H-1′), 5.98 (m, 1H, P—H),6.22 (m, 1H, H-5), 7.0-7.5 (m, 16H, Tr), 8.32 (m, 114, H-6); ¹³P-NMR(161 MHz, DMSO) 9.17, 9.65; LC/MS Scan ES+ 1026 (M+H)⁺, λ_(max)=282.7nm.

To a solution of compound 12 (500 mg, 0.49 mmol) in anhydrous carbontetrachloride (4.9 ml) was added dropwise benzylamine (5 eq, 0.266 ml).The reaction mixture was stirred at room temperature for 3 h and thesolvent removed under reduced pressure. The crude mixture was purifiedon silica gel column chromatography (eluant: stepwise gradient [0-5%] ofmethanol in methylene chloride) to afford compound 13 as a foam (75%).Rf=0.25 (3% MeOH in methylene chloride); ¹H-NMR (400 MHz, DMSO) 0.79 (s,3H, CH₃), 1.13 and 1.06 (2s, 6H, 2CH₃), 3.05 (m, 4H, CH₂OTr, CH₂OS),3.51 (m, 1H, H-3′), 3.69 (s, 6H, 2×OCH₃), 3.87 (m, 3H, CH₂OP, CH₂N,H-3′), 4.08 (m, 2H, H-5′, H-5″), 5.19 and 5.0 (2m, 2H, OH-2′ and OH-3′,D₂O exchangeable), 5.67 (m, 1H, NH, D₂O exchangeable), 5.75 (2s, 1H,H-1′), 6.21 (m, 1H, H-5), 6.7-7.5 (m, 34H, Tr, DMTr, C₆H₅CH₂, H-6);¹³P-NMR (161 MHz, DMSO) 9.84, 9.69; LC/MS Scan ES+ 1132 (M+H)⁺.

Compound 13 can be converted into the phosphoramidate prodrug 10 (B102)following experimental conditions described for the last step inExamples 3 (Procedure A) and in Example 4.

Procedure B

Synthetic Scheme:

B102 is synthesized as a mixture of phosphorous diastereomers in 1:1ratio. Isolated overall yield from NM107 to B102 was 31%, as not all thecoupled material produced was used for deprotection.

Step 1.1:

FW Density Amount Material Grade gmol⁻¹ Quantity gml⁻¹ mol Eq NM107 99%257.2 150 g — 0.583 1 PhB(OH)2 98% 122.1  78 g — 0.639 1.10 Pyridineanhydrous 98% 79.1 2.5 L 0.978 — —

NM 107 was dissolved in pyridine under argon and benzeneboronic acid wasadded. The stirred mixture was heated at reflux for 3 h under argon.Distillation of the azeotrope was then performed, removing 1.2 L(pyridine/water).

T head: 103° C.→113° C. T mixture: 112° C.→116° C.

The mixture was cooled to room temperature and the pyridine wasevaporated under vacuum to get golden oil. The product was stored undervacuum overnight to be used for the next step. A ratio of 97:3product:starting material was observed by 1H-NMR (d6-DMSO). Prior toStep 1.2, the crude was dissolved in 250 mL anhydrous pyridine.Alternatively the following conditions may be used:

-   -   eq benzeneboronic acid    -   5 eq pyridine    -   1.5 eq Na₂SO₄    -   5 mL CH₃CN for 1 g of NM107    -   Heat at reflux for 1 h-1 h 30. Cool to RT. Used for next        reaction.    -   98-99% of conversion by proton NMR        Step 1.2:

FW Density Amount Material Grade gmol⁻¹ Quantity gml⁻¹ mol Eq 2,3-PhB- —343.1 Solution — ~0.583 1 NM107 Phosphonate 3 — 585.7 615 g — 1.049 1.8EDCl•HCl 98% 191.7 570 g — 2.973 5.1 Acetonitrile 98% — 3 L — — —anhydrous Benzylamine 98% 107.2 445 mL 0.98 4.0 7* Carbon 98% 153.8 260mL 1.59 12.6 4* tetrachloride *Extra equivalents of these reagents maybe added (e.g. 15 eq) if required (if P—OH visible by HPLC)

Phosphonate 3 was dissolved in 3 L of acetonitrile under argon. Asolution of 2,3-PhB-NM107 from Step 1.1 was added followed by EDCI.HCl.The mixture was stirred under argon at 41-46° C. for 4 h after whichtime HPLC analysis indicated ˜7:1 ratio of P—H product to NM107. Themixture was cooled to 18° C. and benzylamine was added dropwise followedby carbon tetrachloride. The reaction was slightly exothermic. Analysisby HPLC indicated complete conversion of P—H to phosphoramidate product.Ethyl acetate (1 L) was added to the mixture which was then acidified topH 4 with 3 L of 20% citric acid. The aqueous phase was extracted with2.5 L of ethyl acetate. The organic phases were combined and washed with3 L of 10% citric acid. The organic phase was basified to pH 8 with 5 Lof aqueous sodium bicarbonate (saturated) and washed a second time with2 L of aqueous sodium bicarbonate

(saturated). The organic phase was dried over sodium sulfate, filteredunder vacuum and evaporated to give a yellow foam, 712 g.

The crude residue was dissolved in dichloromethane (1 L) and purified onsilica plug (2.3 Kg of silica). Eluted with: 5 L 4% Methanol/DCM, 2*1 L4%, 3*1 L 5%, 8*250 mL 6%, 4*250 mL 7%, 9*1 L 7%. Evaporation of therelevant fractions gave 254 g (HPLC purity: 98.5%, yield: 52%) and 73 g(HPLC purity: 87.6%, yield: 13%) of phosphoramidate 4.

Step 2:

FW Density Amount Material Grade Gmol⁻¹ Quantity gml⁻¹ mol EqPhosphoramidate 4 — 828.9 246 g — 0.291 1 AcCl 99%  78.5 62.6 mL 1.1051.049 3.0* EtOH anhydrous 98% — 3.5 L* — — — *Subsequently 2.0 eq AcCland 1:10 w/v 4:EtOH ratio were used.

Phosphoramidate 4 was dissolved in anhydrous ethanol and acetyl chloridewas added (exothermic: 18° C. to 27° C.) to the reaction mixture, underargon. The mixture was stirred at 60° C. under argon. After 30 min, HPLCanalysis indicated complete conversion of the phosphoramidate 4 todeprotected product 5. The mixture was cooled to 25° C. and solid sodiumbicarbonate (1.04 Kg) was added in several portions (foaming, pH˜5.5-6). The mixture was filtered through Celite and washed with twovolumes of ethanol. The filtrate was evaporated under vacuum at 35° C.The residue was triturated with TBME (3 L) for 1 h and then filtered toremove the trityl by-product. The solid obtained was dried under vacuumto give 185 g with 93% purity at 254 nm by HPLC.

If required, any residual benzeneboronic acid may be removed from theproduct by dissolution in water and treatment with Amberlite IRA-743resin.

The following alternative reaction conditions (to avoid the possibilityof acylating 4) are possible:

-   -   2.0 eq AcCl in EtOH 1:10 v:v to generate HCl and consume all        AcCl (exothermic)    -   Phosphoramidate 4 in EtOH (to make 1:10 w:v total volume EtOH)    -   Add HCl/EtOH solution to reaction mixture at 20° C.    -   60° C. under argon, 30-45 min        The crude was purified by reverse-phase chromatography (1.5 Kg        of prepared Bakerbond 40 μm C-18 RP-silica—washed with 100%        acetonitrile gradient to 100% H₂O). The crude was dissolved in        acetonitrile (58 mL), H₂O (164 mL) and saturated aqueous sodium        bicarbonate solution (170 mL).        Elution under gentle vacuum with a stepwise gradient of 3%        MeCN/H₂O, 10%, 15%, 25% (pure product eluted) and evaporation of        the relevant fractions gave 106 g B102 (62% yield) with 98.6%        purity at 254 nm by HPLC.        Typical analytical data is shown below:

B102: C₂₄H₃₅N₄O₉PS 586.59 gmoF⁻¹

HPLC AUC (Method Test 20): 98.9%@254 nm, Rt 3.34 min

m/z (ESI+): 587.12 [M+H]⁺ 100%; 1173.62 [2M+H]⁺ 80%

v_(max) (KBr disc) (cm⁻¹): 3343.1 br (O—H, N—H); 1647.2 br (C═O base,thioester)

KF: 2.02% H₂O content

Specific Rotation: [α]_(D) ²⁰+55.011 (c. 10.492 mg cm⁻³ in DMSO)

Elemental analysis: Calculated: C, 49.14%; H, 6.01%; N, 9.55%; S, 5.47%;P, 5.28%;

Found: C, 48.74%; H, 5.83%; N, 9.41%; S, 5.81%; P, 5.33%.

NMR: Analyzed using ¹H, ¹³C, ³¹P, COSY, DEPT, HSQC and HMBC experiments.¹14 NMR δ_(H) (400 MHz, d6-DMSO): 0.94 (3H, d, J=1.8 Hz, CH₃), 1.11 (6H,s, (CH₃)₂C), 3.04 (2H, m, J=6.4 Hz, CH₂S), 3.44 (2H, d, J=5.0 Hz,CH₂OH), 3.60 (1H, br-m, H-3′), 3.82-4.01 (5H, m, H-4′, CH₂O, CH₂Ph),4.07-4.12 (1H, m, H-5′), 4.13-4.24 (1H, m, H-5″), 4.94 (1H, t, J 5.0 Hz,CH₂OH), 5.07 (1H, d, J 1.8 Hz, OH-2′), 5.26 (1H, t, J 6.8 Hz, OH-3′),5.64-5.76 (1H, m, P—N—H), 5.69, 5.70 (1H, 2×d, 2×J 7.6 Hz, H-5), 5.93(1H, br-s, H-1′), 7.13-7.20 (2H, 2×br-s, NH₂), 7.20-7.25 (1H, m, Ar—H),7.28-7.35 (4H, m, 4×Ar—H), 7.53, 7.57 (1H, 2×d, J 7.6 Hz, H-6)

¹³C NMR δ_(C) (100 MHz, d6-DMSO): 19.81 (CH₃), 21.79 (C(CH₃)₂), 28.17,28.24 (CH₂S), 44.18 (PhCH₂), 51.62 (C(CH₃)₂), 63.74, 63.79 (CH₂O),64.21, 64.51 (C-5′), 68.29 (CH₂OH), 72.41, 72.57 (C-3′), 77.80, 77.85(C-2′), 79.47, (C-4′), 91.66, (C-1′), 93.82 (C-5), 126.68, 127.09,128.08, 128.09 (5×Ar—C), 140.34, 140.38, 140.40 (Ar—C_(ipso), C-6),155.12, 165.21 (C-2, C-4), 203.85 (C═OS) ³¹P NMR δ_(P) (162 MHz,d6-DMSO): 9.71, 9.91 (1P, 2×s, ratio 1.00:1.07)

Synthetic Procedure A can be used for synthesizing nucleoside prodrugssuch as B 102. Protection of the 2′ and 3′ hydroxyl groups as well asthe amino group that may be present on the nucleoside base is preferred.In Strategy A, the 2′ and 3′ hydroxyl groups are protected, e.g., as theacetonide derivative and the amino group is protected, e.g., as thedi-methoxytrityl derivative. Hydrolysis of the acteonide after thecoupling of the nucleoside with the SATE intermediate is carried outusing an acid such as TFA. This hydrolysis procedure can potentiallyproduce by-products and give low yields, and the di-methoxytritylchloride is disadvantageously expensive. Synthetic Procedure B, below,can overcome the such difficulties. An acid, such as a boronic acid,such as phenyl boronic acid is used to protect the 2′ and 3′ hydroxylgroups on the sugar moiety. Coupling the nucleoside phenyl boronatederivative with the SATE intermediate can give good yield, and thephenyl boronate deprotection conveniently takes place during the work-upof the reaction mixture, on washing with an acid such as an aqueouscitric acid solution. The final removal of a protecting group such as atrityl group (on the Sate moiety) is mildly carried out using an organicsolvent system, such as an acetyl chloride/ethanol mixture. Thisdeprotection reaction can be consistently reproducible, scalable andgave significantly high yield.

Example 3 Preparation of B299, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of 2′-C-methylguanosine

Procedure A

Synthetic Scheme:

2′-C-methylguanosine (NM108) (3 g, 10.10 mmol) and compound 5 [for thesynthesis of 5 See Example 2] (6.48 g, 11.10 mmol) were coevaporatedtogether with anhydrous pyridine and dissolved in this solvent (152 mL).Pivaloyl chloride (2.48 mL, 20.18 mmol) was added dropwise at −15° C.and the solution was stirred at the same temperature for 2 h. Thereaction mixture was diluted with methylene chloride and neutralizedwith an aqueous solution of ammonium chloride (NH₄Cl 0.5M). Afterextraction with methylene chloride/aq NH₄Cl 0.5M, the organic phaseswere combined, dried over Na₂SO₄ evaporated under reduce pressure (bathtemperature not exceeding 30° C.) and coevaporated twice with toluene.The crude mixture was purified on silica gel flash column chromatography(eluant: stepwise gradient [0-10%] of methanol in methylenechloride+0.2% acetic acid) to afford the desired product 6 (2.5 g, 32%).R_(f)=0.34 (15% MeOH in CH₂Cl₂); ¹H-NMR (400 MHz, DMSO-d₆ 0.80 (s, 3H,CH₃), 1.13 (s, 6H, 2×CH₃), 3.04 (m, 2H, CH₂OTr), 3.14 (m, 2H, CH₂S),3.97-4.08 (m, 4H, H-3′, H-4′, CH₂OP), 4.28-4.38 (m, 2H, H-5′, H-5″),5.10-5.35 (m, 2H, OH-2′, OH-3′, D₂O exchangeable), 5.77 (s, 1H, H-1′),6.52 (bs, 2H, NH₂, D₂O exchangeable), 7.11-7.42 (m, 15H, Tr), 7.75 (s,1H, H-8), 10.67 (bs, 1H, NH, D₂O exchangeable); ¹³P-NMR (161 MHz,DMSO-d₆) 9.47, 9.20; LC/MS Scan ES+ 764 (M+H)⁺, Scan ES− 762 (M−H)⁻.

To a solution of compound 6 (2.5 g, 3.27 mmol) in anhydrous carbontetrachloride (33 mL) was added dropwise benzylamine (5 eq, 1.79 mL).The reaction mixture was stirred at room temperature for 1 h andevaporated under reduced pressure (bath temperature not exceeding 30°C.). The crude mixture was purified on silica gel flash columnchromatography (eluant: stepwise gradient [0-10%] of methanol inmethylene chloride) to give compound 7 as a white foam (2.9 g,quantitative yield). Rf=0.27 (10% MeOH in methylene chloride); ¹H-NMR(400 MHz, DMSO-d₆) 0.81 (s, 3H, CH₃), 1.10 (s, 6H, 2×CH₃), 2.99-3.08 (m,4H, CH₂OTr, CH₂S), 3.87-4.30 (m, 8H, H-3′, H-4′, H-5′, H-5″ CH₂OP,NCH₂Ph), 5.66 (m, 1H, NH, D₂O exchangeable), 5.76 (s, 1H, H-1′), 6.60(bs, 2H, NH₂, D₂O exchangeable), 7.17-7.39 (m, 20H, Tr, C₆H₅CH₂), 7.77(s, 1H, H-8); ¹³P-NMR (161 MHz, DMSO-d₆) 9.93, 9.78; LC/MS Scan ES+ 869(M+H)⁺, Scan ES− 867 (M−H)⁻.

Compound 7 (2.84 g, 3.27 mmol) was dissolved in a mixture oftrifluoroacetic acid (1.1 mL) and methylene chloride (11.4 mL). Thereaction mixture was stirred 0.5 h at room temperature. The solution wasdiluted with ethanol, evaporated under reduce pressure (bath temperaturenot exceeding 30° C.) and coevaporated twice with toluene. The crudemixture was purified on silica gel flash column chromatography (eluant:stepwise gradient [0-30%] of methanol in methylene chloride) and then,on reverse phase column chromatography (eluant: stepwise gradient[0-100%] of acetonitrile in water) to give the desired product 8 (B299)(1:1 mixture of diastereoisomers according to ³¹P-NMR, 800 mg, 39%)which was lyophilized from a mixture of dioxan/water. Rf=0.57 (20% MeOHin methylene chloride); ¹H-NMR (400 MHz, DMSO-d6) 0.82 (s, 3H, CH₃),1.09 (s, 6H, 2×CH₃), 3.01 (m, 2H, CH₂S), 3.42 (d, 2H, CH₂OH, J=8.0 Hz),3.81-4.00 (m, 6H, H-3′, H-4′ CH₂OP, NCH₂Ph), 4.11-4.27 (m, 2H, H-5′,H-5″), 4.92 (t, 1H, CH₂OH, J=8.0 Hz, D₂O exchangeable), 5.16 (s, 1H,OH-2′, D₂O exchangeable), 5.40 (m, 1H, OH-3′, D₂O exchangeable), 5.64(m, 1H, NH, D₂O exchangeable), 5.75 (s, 1H, H-1′), 6.50 (bs, 2H, NH₂.D₂O exchangeable), 7.19-7.32 (m, 5H, PhH), 7.77 (s, 1H, H-8), 10.61 (bs,1H, NH, D₂O exchangeable); ¹³P-NMR (161 MHz, DMSO-d6) 9.91, 9.78; HPLCt_(R)=3.67 min (0-100% acetonitrile over a period of 8 min),λ_(max)=251.3; LC/MS Scan ES+ 627 (M+H)⁺, Scan ES− 625 (M−H)⁻.

Example 4 Preparation of B208, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of 2′-C-methylthymidine

Synthetic Scheme:

2′-C-Methylthymidine (NM105) (700 mg, 2.57 mmol) and 5 [for thesynthesis of 5, See Example 2] (1.1 eq, 1.6 g) were coevaporatedtogether with anhydrous pyridine and dissolved in this solvent (40 ml).Pivaloyl chloride (2.0 eq, 0.633 ml) was added dropwise at −15° C. andthe solution stirred at this temperature for 1 h 30. The reactionmixture was diluted with methylene chloride and neutralized with anaqueous solution of ammonium chloride (NH₄Cl 0.5M). After extractionwith methylene chloride/aq NH₄Cl 0.5M, the organic phases were combined,evaporated under reduced pressure (bath temperature not exceeding 30°C.) and coevaporated with toluene. The crude mixture was purified onsilica gel column chromatography (eluant: stepwise gradient [0-10%] ofmethanol in methylene chloride+0.2% acetic acid) to afford the desiredproduct 6 which was coevaporated with toluene to give a white foam (942mg, 50%). R_(f)=0.56 (15% MeOH in CH₂Cl₂); ¹H-NMR (400 MHz, DMSO) 1.00(s, 3H, CH₃), 1.13 (s, 6H, 2CH₃), 1.77 (s, 3H, CH₃), 3.16 (m, 2H, CH₂S),3.32 (m, 2H, CH₂OTr), 3.6 (m, 1H, H-3′), 3.9 (m, 1H, H-4′), 4.0 (m, 2H,CH₂OP), 4.2-4.3 (m, 2H, H-5′, H-5″), 5.21 (s, 1H, OH-2′, D₂Oexchangeable), 5.40 (t, 1H, OH-3′, D₂O exchangeable), 5.83 (s, 1H,H-1′), 6.0 (s, 1H, P—H), 7.0-7.5 (m, 16H, Tr, H-6); ¹³P-NMR (161 MHz,DMSO) 9.29, 9.68; LC/MS Scan ES+ 761 (M+Na)⁺.

To a solution of compound 6 (920 mg, 1.25 mmol) in anhydrous carbontetrachloride (13 ml) was added dropwise benzylamine (10 eq, 1.4 ml).The reaction mixture was stirred at room temperature for 2 h. A whiteprecipitate appeared. The solution was diluted with methylene chlorideand neutralized with an aqueous solution of hydrogen chloride (HCl 1M).After successive extractions with CH₂Cl₂/HCl 1M and CH₂Cl₂/aq NaHCO₃,the organic phases were combined, dried over Na₂SO₄, filtered andevaporated to dryness. The crude mixture was purified on silica gelcolumn chromatography (eluant: stepwise gradient [0-10%] of methanol inmethylene chloride) to give 7 as a white foam (875 mg, 83%). R_(f)=0.56(15% MeOH in CH₂Cl₂); ¹H-NMR (400 MHz, DMSO) 0.99 (s, 3H, CH₃), 1.12 (s,6H, 2CH₃), 1.75 (s, 3H, CH₃), 3.04 (m, 4H, CH₂OTr, CH₂S), 3.69 (m, 1H,H-3′), 3.8-4.0 (m, 5H, CH₂OP, CH₂N, H-4′), 4.0-4.2 (m, 2H, H-5′, H-5″),5.17 (s, 1H, OH-2′, D₂O exchangeable), 5.3 (m, 1H, OH-3′, D₂Oexchangeable), 5.7 (m, 1H, NH, D₂O exchangeable), 5.82 (s, 1H, H-1′),7.1-7.5 (m, 21H, Tr, C₆H₅CH₂, H-6); ¹³P-NMR (161 MHz, DMSO) 9.95, 9.86;HPLC t_(R)=7.91 min (0-100% acetonitrile over a period of 8 min),λ_(max)=266.7 nm; LC/MS Scan ES+ 866 (M+Na)⁺.

Finally, compound 7 (860 mg, 1.02 mmol) was dissolved in a mixture ofmethylene chloride (15 ml) and trifluoroacetic acid (0.51 ml). Thereaction mixture was stirred at room temperature for 2 h, then dilutedwith toluene. The volatiles were evaporated under reduced pressure andcoevaporated with ethanol. The crude mixture was purified by silica gelcolumn chromatography (eluant: stepwise gradient of methanol [0-10%] inmethylene chloride), followed by a purification on reverse phasechromatography (eluant: stepwise gradient of acetonitrile [0-50%] inwater), to give the desired product 8 (B208) (257 mg, 42%). Rf=0.31 (10%MeOH in methylene chloride); ¹H-NMR (400 MHz, DMSO-d6) 0.99 (s, 3H,CH₃), 1.10 (s, 6H, 2×CH₃), 1.75 (s, 3H, CH₃), 3.0 (m, 2H, CH₂S), 3.42(d, 2H, CH₂OH), 3.7 (m, 1H, H-3′), 3.8-4.0 (stack, 5H, CH₂OP, NCH₂Ph,H-4′), 4.0-4.3 (m, 2H, H-5′ and H-5″), 4.9 (m, 1H, CH₂OH, D₂Oexchangeable), 5.17 (s, 1H, OH-2′, D₂O exchangeable), 5.3 (m, 1H, OH-3′,D₂O exchangeable), 5.7 (m, 1H, NH, D₂O exchangeable), 5.81 (s, 1H,H-1′), 7.2-7.4 (stack, 6H, PhH, H-6); ¹³P-NMR (161 MHz, DMSO-d6) 9.84,9.90; HPLC t_(R)=4.98 min (0-100% acetonitrile over a period of 8 min),λ_(max)=269.0 nm; LC/MS Scan ES+ 602 (M+H)⁺.

Example 5 Preparation of B261, the Hydroxy-tBuSATEN-benzylphosphonamidate Derivative of PMEA

Procedure A

Synthesis of Intermediate 4:

A 500 mL triple-neck flask fitted with a condenser was charged with PMEA(2.00 g, 7.25 mmol), CH2Cl2 (121 mL) and DMF (617 μL, 7.98 mmol). Theresulting slurry was vigorously stirred and oxalyl chloride (2.21 mL,25.4 mmol) was added dropwise at 0° C. over 10 min (gas evolution). Theslurry turned to a yellow solution (10 min) before turning turbid (10min). This was further stirred for 3 h under reflux and turned to awhite, thick slurry. The products were schlenk-dried 1 h in situ byevaporation of all volatiles under reduced pressure, at roomtemperature. The resulting yellow solid could then be partiallydissolved in CH2Cl2 (121 mL), and pyridine (1.17 mL, 14.5 mmol) wasadded dropwise at 0° C. over 10 min. The white suspension turned to ablue solution, which was cooled to −78° C. A solution of alcohol 3 [forthe synthesis of 3, See Example 1] (3.480 g, 7.25 mmol) andtriethylamine (6.37 mL, 45.7 mmol) in CH2Cl2 (72 mL) was then addedslowly (ca. 45 min), dropwise along the internal wall, and the reactionwas stirred 10 h at −78° C. Benzylamine (2.37 mL, 21.7 mmol) was thenadded dropwise at −78° C. and the solution was left stirring warming tor.t. over 1 h. NaHCO₃ (aq. sat., 200 mL) was poured over the reactionand the layers separated. The aqueous phase was extracted with CH2Cl2(2×100 mL) and the combined organic extracts were dried with brine (50mL) and Na₂SO₄. The solution was filtered and concentrated to afford ca.6.5 g of crude yellow syrup. Purification by flash column chromatography(SiO2, Ø=3.5 cm, H=11 cm) eluting with 4→8→12% MeOH in CH2Cl2 (1% Et3N)afforded 3.70 g of a yellow foam (0.15<Rf<0.30, 10% MeOH in CH2Cl2) thatwere submitted to a second purification by flash column chromatography(SiO2, Ø=3.5 cm, H=12 cm) eluting with 4→6% MeOH in CH2Cl2 (1% Et3N) toafford 2.67 g of a yellow foam (0.16<Rf<0.25, 10% MeOH in CH2Cl2). Thiswas submitted to a third purification by flash column chromatography(SiO2, Ø=3.5 cm, H=12 cm) eluting with 4→6% MeOH in CH2Cl2 (1% Et3N) toproduce 165 mg of phosphonamidate 4 (ca. 2.7%) as a white foam and 1.75g of mixed compounds. These were submitted to a last purification byflash column chromatography (SiO2, Ø=3.5 cm, H=12 cm) eluting with 4→6%MeOH in CH2Cl2 (1% Et3N) afforded 353 mg of phosphonamidate 4 (ca. 5.9%)as a white foam. Total yield: 8.6%. Rf=0.21 (6% MeOH in CH2Cl2); 1H-NMR(300 MHz, CDCl3) 1.13 (s, 6H, 2CH₃), 3.02-3.10 (m, 2H, CH2S), 3.59 (t, J7.5, 2H, CH₂), 3.58 (s, 6H, 2×OCH₃), 3.73 (t, J 7.1, 2H, CH2), 3.88-4.09(stacks, 4H, 2×CH2), 4.21 (t, J 7.0, 2H, CH2O), 5.50 (br s, 2H, NH2),6.67-6.78 (m, 4H, PhH), 7.04-7.38 (stack, 9H, PhH), 7.72 (s, 1H), 8.22(s, 1H); 31P-NMR (121 MHz, CDCl3) 25.0; m/z (FAB+) 825 (1), 303 (100);HRMS 825.3171 ([M+H]⁺. C43H50O7N6PS requires 825.3199).

Synthesis of Compound 5 (B261):

Dichloroacetic acid (20% solution in CH2Cl2, ca. 140 drops) was addeddropwise to a solution of ether 4 (353 mg, 0.43 mmol) in CH2Cl2 (4.3 mL)at 0° C. and this was stirred for 55 min. Solid NaHCO3 (ca. 1.5 g) wasthen added and the slurry was stirred for 10 min before filtration andevaporation. Purification by flash column chromatography (SiO2, Ø=1.5cm, H=10 cm) eluting with 4% 10% MeOH in CH2Cl2 afforded purephosphonamidate 5 (130 mg after lyophilization in THF/H2O and 3 day-stayin P2O5 desiccator, 58%). This reaction was also performed on 165 mg ofether 4 to produce 51 mg of phosphonamidate 5 (B261, 49%). Rf=0.20 (10%MeOH in CH₂Cl₂); 1H-NMR (300 MHz, DMSO-d6) 1.10 (s, 6H, 2×CH3), 2.80 (t,J 7.0, 2H, CH2S), 3.43 (d, J 5.5, 2H, CH2OH), 3.69 (A of AB, J 4.8, 1H,1×CH2P), 3.71 (B of AB, J 4.8, 1H, 1×CH2P), 3.75-3.88 (stacks, 4H, CH2O,NCH2), 3.88-4.07 (m, 2H, NCH2Ph), 4.30 (t, J 7.0, 2H, CH2O), 4.97 (t, J6.1, 1H, OH), 5.31-5.42 (m, 1H, NH), 7.16-7.32 (stack, 7H, PhH, NH2),8.09 (s, 1H), 8.13 (s, 1H); 13C-NMR (75 MHz, DMSO-d6) 21.8 (2×CH3), 28.4and 28.5 (CH2, CH2S), 42.4 (CH2, NCH2), 43.3 (CH2, NCH2), 51.7 (quat. C,C(CH3)2), 61.7 and 61.8 (CH2, CH2O), 64.6 (CH2, CH2O), 68.4 (CH2, CH2O),118.5 (quat. C), [126.5 (CH, Ph), 127.0 (CH, Ph), 128.0 (CH, Ph), someoverlap], 140.5 and 140.6 (quat. C), 141.0 (CH), 149.4 (quat. C), 152.3(CH), 155.9 (quat. C), 203.9 (quat. C═O); 31P-NMR (121 MHz, DMSO-d6)25.9; m/z (FAB+) 161 (32), 256 (42), 523 (100); HRMS 523.1899 ([M+H]+.C22H32O5N6PS requires 523.1892); HPLC(C18, flow: 0.5 mL/min, solutionA=TEAC 20 mM, solution B=20% TEAC 20 mM): tR=5.04 min (60% A in B),tR=27.24 min (t=0→10 min: 100% A; t=10→30 min: 0→50% B in A; t=30→35min: 50→100% B in A); UV (EtOH 95%) λ_(max)=205 (εmax 23900), λmin=228(εmin 5400).

Procedure B Improved Preparation of the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative (B261, Compound 5) of PMEA

Synthetic Scheme:

Step 1: Synthesis of Intermediate A

To the suspension of PMEA (2 g, 7.3 mmol) in 120 mL of DCM (anhydrous)was added DMF (640 mg, 1.2 eq), followed with oxalyl chloride (2.3 mL,3.5 eq) at room temperature. The mixture was heated to reflux for 1.5hrs to give a thick yellow suspension. The mixture was concentrated todryness through rotarvap to give the crude intermediate 2 as pale yellowsolid. LCMS analysis of the aliquot of intermediate 2 in methanolicsolution confirmed the structure of the product in good purity.

Step 2: Synthesis of Intermediate B:

The crude intermediate A (7.33 mmol) was suspended into 100 mL ofanhydrous DCM. The suspension was cooled to 0° C. To this was addedpyridine (1.2 mL, 14.6 mmol, 2 eq) at 0° C. After the addition, the paleyellow suspension turned to a golden colored clear solution. Thissolution was cooled to −32° C. with ACN/dry ice bath. To this was addeda solution of a (3.52 g, 7.33 mmol, 1 eq) in 70 mL of anhydrous DCM thatcontained triethylamine (6.3 mL, 44 mmol, 6 eq) drop-wise. The internalreaction temperature was maintained between −35° C.˜−30° C. during theaddition. The bright golden colored solution turned to a green coloredsolution with some precipitate crashing out from the solution during theaddition. The precipitate was presumably the triethylamine HCl salt. Ittook 20 minutes to complete the addition. After the addition, themixture was stirred at −30° C.˜−10° C. for 1 hr. The reaction mixturewas cooled back to −20° C. To this was added benzylamine (2.4 mL, 22mmol, 3 eq). The mixture was stirred at −20° C. for 10 minutes. To thereaction mixture was added saturated NaHCO₃/H₂O and the mixture wasstirred for 2 minutes. The DCM layer was separated, dried with Na₂SO₄and concentrated to dryness to give the crude intermediate a as yellowviscous oil. HPLC analysis of the crude intermediate gave 62% purity at272 nm.

Step 3: Synthesis of Intermediate 4:

The crude intermediate it (7.33 mmol) as viscous pale yellow oil wasdissolved into 200 mL of MeOH. The reaction mixture was refluxedovernight. HPLC analysis of the reaction mixture indicated the completeconversion of the amidine to amine. [The retention time of amidine(RT=5.92 min) is very close to the amine (RT=5.98 min) by the currentin-house HPLC method!] The mixture was cooled to RT and filtered. Thefiltrate was concentrated to dryness by rotar-vap. The obtained crudeproduct was purified through silica gel column chromatography (120 gsilica gel combiflash column was used, 3-8% of MeOH in DCM as theeluent) to give 3.1 g pure product 4 as white foam in 51% isolated yieldfrom 2 g of PMEA. 1H-NMR of the obtained 4 was consistent with thedesired structure. HPLC analysis of the obtained 4 gave 96% purity(AUC).

Step 4: Synthesis of B261 (Compound 5.)

Intermediate 4 (300 mg, 0.36 mmol) was dissolved into EtOH (anhydrous, 5mL). To this was added acetyl chloride (43 mg, 1.5 eq) in one portion atroom temperature. The reaction should be operated in a closed reactionflask to avoid the loss of HCl gas. The reaction mixture was stirred atRT for 30 min. To this was added solid NaHCO₃ and the mixture wasstirred for 15 min. pH of the reaction mixture was found to be around7-8. The mixture was filtered and the filtrate was concentrated todryness. The crude product was purified through silica gel columnchromatography (5-10% MeOH in DCM as the eluent) to give 163 mg of 5 asclear viscous oil in 86% yield. 1H-NMR of the obtained product wasconsistent with the desired structure. HPLC analysis of the obtainedproduct gave 97.4% purity (AUC).

Example 6 Preparation of B263, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of 2′-C-methyladenosine

Synthetic Scheme:

The pronucleotide B263 (94 mg, 6% overall yield) has been synthesizedfrom its parent nucleoside 2′-C-methyl-6-NH-dimethoxytrityl-adenosine(1.59 g, 2.73 mmol) following a similar procedure than the one describedfor the synthesis of the pronucleotide prepared in the Example 2(Procedure A, Strategy b), and isolated as a white lyophilized powder.¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 0.80 (s, 3H), 0.97-0.98 (d, J=4.26 Hz,6H), 3.02 (m, 2H), 3.34-3.35 (m, 2H), 3.76-3.96 (m, 4H), 4.03-4.05 (m,2H), 4.15-4.17 (m, 2H), 4.76-4.79 (m, 1H), 5.32 (s, 1H), 5.34-5.36 (m,1H), 5.45-5.55 (m, 1H), 5.93 (s, 1H), 7.1-7.4 (m, 7H), 8.14 (s, 1H),8.21 (1H); ³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm) 9.75 and 9.86 (2s); ScanES⁺ 611 (M+H)⁺, λ_(max)=258 nm; HPLC (0-100% ACN over a period of 8 min)t_(R)=4.79 min λ_(max)=260.8 nm.

Example 7 Preparation of B229, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of 2′-C-methyluridine

Reagents and Conditions: (i) pTsOH.H₂O, CH(OEt)₃, acetone, r.t.; (ii)PivCl, pyr, r.t; (iii) Benzylamine, CCl₄, rt; (iv) aq. 90% TFA, DCM,r.t*

The pronucleotide 6 (446 mg, 0.76 mmol, overall yield 9% over 4 steps)has been synthesized from its nucleoside parent 1 following a similarprocedure described for the synthesis of the pronucleotide prepared inExample 2, Strategy A.

B229 6.

¹H NMR (400 MHz, DMSO-d₆): δ 0.98 (s, 3H, CH₃); 1.10 (s, 6H, 2×CH₃);3.03 (m, 2H, CH₂S); 3.41 (m, 2H, CH ₂OH, J=5.6 Hz); 3.61 (m, 1H, H-3′);3.8-4.0 and 4.05-4.25 (stacks, 5H, NCH ₂Ph, H-4′, H-5′ and H-5″);4.05-4.25 (2×1H, 2×m, CH ₂OP); 4.91 (t, 1H, 3′-OH, D₂O exchangeable,J=5.62 Hz); 5.20 (br-s, 1H, 2′-OH, D₂O exchangeable); 5.39 (a-t, 1H,CH₂OH, D₂O exchangeable, J=7.32 Hz); 5.52 (m, 1H, H-5); 5.65 (m, 1H,PhNH, D₂O exchangeable); 5.8 (br-s, 1H, H-1′); 7.2-7.32 (m, 5H, ArH);7.55 (a-dd, 1H, H-6); 11.37 (br-s, 1H, NH, D₂O exchangeable).

³¹P NMR (161.8 MHz, DMSO-d₆): δ 9.73 and 9.98 (ratio of signals byintegration of 52:48) m/z (ES+) 588.11 (M+H)⁺.

HPLC (Method 20): chemical purity 99.2%, 3.48 mins.

CHN analysis:—Found: C, 49.29; H, 5.95; N, 6.88; P, 5.16; C₂₄H₃₄N₃O₁₀PSrequires C, 49.06; H, 5.83; N, 7.15; P, 5.46.

[α]_(D) ²³+26.3 (c, 0.571 in H₂O).

v_(max) (KBr): 3373 (br, NH and OH), 1682 (C═O).

Example 8 Preparation of B186, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of 2′-C-methylinosine

Synthetic Scheme:

The pronucleotide B186 (314 mg, 8% overall yield) has been synthesizedfrom its parent nucleoside 2′,3′-O-isopropylidene-2′-C-methyl-inosine(2.0 g, 6.26 mmol) following a similar procedure than the one describedfor the synthesis of the pronucleotide prepared in the Example 2(Procedure A, Strategy a), and isolated as a white lyophilized powder.¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 0.79 (s, 3H), 1.09 (s, 6H), 3.01-3.04(t, J=6.53 Hz, 2H), 3.42 (s, 2H), 3.84-3.91 (m, 2H), 3.94-4.03 (m, 3H),4.05-4.09 (m, 1H), 4.15-4.26 (m, 2H), 4.92 (s, 1H), 5.36 (s, 1H), 5.43(t, J=6.54 Hz, 1H), 5.62-5.71 (m, 1H), 5.94 (s, 1H), 7.18-7.22 (m, 1H),7.25-7.30 (m, 4H), 8.08 (s, 1H), 8.10 (s, 1H), 12.15 (brs, 1H); ³¹P NMR(DMSO-d₆, 162 MHz) δ (ppm) 9.76-9.90 (2s); Scan ES⁺ 612 (M+H)⁺,λ_(max)=240.7 nm; HPLC (0-100% ACN over a period of 8 min) t_(R)=4.72min λ_(max)=243.1 nm.

Example 9 Preparation of B396, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of9-[2-C-methyl-β-ribofuranosyl]-6-chloropurine

Synthetic Scheme:

The pronucleotide B396 (75 mg, 10% overall yield) has been synthesizedfrom its parent nucleoside 9-[2-C-methyl-β-ribofuranosyl]-6-chloropurine(571 mg, 1.90 mmol) following a similar procedure than the one describedfor the synthesis of the pronucleotide prepared in the Example 4 andisolated as a white lyophilized powder. ¹H NMR (DMSO-d₆, 400 MHz) δ(ppm) 0.82 (d, J=2.63 Hz, 3H), 1.07 (s, 6H), 3.02 (m, 2H), 3.40-3.41 (q,J=3.36 Hz and J=1.89 Hz, 2H), 3.85-3.98 (m, 4H), 4.12 (s, 2H), 4.25 (m,2H), 4.89-4.90 (m, 1H), 5.47 (s, 1H), 5.50 (s, 1H), 5.62-5.70 (m, 1H),6.10 (d, J=1.23 Hz, 1H), 7.17-7.29 (m, 5H), 8.76 (s, 1H), 8.82 (s, 1H);³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm) 9.91 and 9.79 (2s); Scan ES⁺ 630(M+H)⁺, λ_(max)=260 nm; HPLC (0-100% ACN over a period of 8 min)t_(R)=4.42 min λ_(max)=265 nm.

Example 10 Preparation of B307, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of2′,3′-O-carbonate-2′-C-methylguanosine

Synthetic Scheme:

N-Benzylaminyl-2′,3′-O-carbonate-2′-C-methylguanosin-5′-yl-O-(triphenylmethyloxy-tert-butyl-S-acyl-2-thioethyl)phosphate(C1)

Compound B2 [See, Compound 7, Example 3, Procedure A] (250 mg, 0.288mmol) was dissolved in dimethylformamide (3.5 mL) and treated with1,1-carbonyldiimidazole (186.60 mg, 1.15 mmol). The mixture was stirredat room temperature for 4 h 30 and concentrated under reduced pressure(bath temperature not exceeding 30° C.). The crude residue was subjectedto silica gel chromatography, eluting with a gradient 0-10% methanol indichloromethane, to give C1 as a colorless oil. (68 mg, 26%). CompoundC1: ¹H NMR (400 MHz, DMSO-d₆) δ 10.80 (ls, 1H, NH), 7.80 (s, 1H, H-8),7.33-7.18 (m, 20H, 4C₆H₅), 6.66 (sl, 2H, NH₂), 6.30 (s, 1H, H-1′), 5.78(m, 1H, PNH), 5.22 (m, 1H, H-3′), 4.47-4.30 (m, 2H, H-4′ and H-5′ a),4.20-4.05 (m, 1H, H-5′ b), 3.99-3.87 (m, 4H, CH₂O and CH₂N), 3.10-3.03(m, 4H, CH₂S and CH₂OTr), 1.27 (s, 3H, CH₃), 1.11 (s, 6H, 2CH₃). ³¹P NMR(162 MHz, DMSO-d₆) δ 10.42 (s), 10.18 (s). LR LC/MS (M+H⁺) 895.4 (5.57min). UV: λ_(max)=253 nm.

N-Benzylaminyl-O-(hydroxy-tert-butyl-S-acyl-2-thioethyl)-2′,3′-O-carbonate-2′-C-methylguanosin-5′-ylphosphate B307 (Compound C2)

Compound C1 (65 mg, 0.073 mmol) was dissolved in dichloromethane (260μL) and treated with TFA (26 μL). The mixture was stirred at roomtemperature for 15 min, then diluted with ethanol, evaporated to dryness(bath temperature not exceeding 30° C.) and coevaporated with toluene.The resulting residue was purified by reverse phase (C 18) silica gelcolumn chromatography eluting with a gradient 0-100% acetonitrile inwater and lyophilised from a mixture of water/dioxane to give B307(Compound C2) (34 mg, 72%, white lyophilised powder). B307 (CompoundC2): ¹H NMR (400 MHz, DMSO-d₆) δ10.84 (ls, 1H, NH), 7.80 (s, 1H, H-8),7.32-7.20 (m, 5H, C₆H₅), 6.69 (ls, 2H, NH₂), 6.30 (s, 1H, H-1′), 5.77(m, 1H, PNH), 5.25 (d, 1H, H-3′, J_(3′4′)=20.0 Hz), 4.92 (ls, 1H, OH),4.50-4.41 (s, 2H, CH₂OH), 3.03 (t, 2H, CH₂S, J_(CH2S-CH2O)=8.0 Hz), 1.30(s, 3H, CH₃), 1.10 (s, 3H, CH₃), 1.08 (s, 3H, CH₃). ¹³C NMR (100 MHz,DMSO-d₆): δ 204.4 (C═O), 154.5 (C-4), 153.1 (C-2), 150.7 (C-6), 140.9(C₆H₅), 135.6 (C-8), 128.7-127.3 (5C, C₆H₅), 117.0 (C-5), 89.7 (C-1′),83.7 and 83.6 (2C, C-2′ and C-3′), 81.8 (C-4′), 68.8 (CH₂OH), 65.1(CH₂O), 64.5 (C-5′), 52.2 (C(CH₃)₂CH₂OH), 44.7 (CH₂N), 28.7 (CH₂S), 22.3(2C, 2CH₃), 18.3 (CH₃). ³¹P NMR (162 MHz, DMSO-d₆) δ 10.39 (s), 10.15(s). LR LC/MS (2M+H⁺) 1305.4 (M+H⁺) 653.2 (2M−H⁻) 1303.8 (M−H⁻) 651.4(5.57 min). HRFAB-MS C₂₆H₃₄O₁₀N₆PS (M+H⁺) calculated 653.1795. found653.1819. UV: λ_(max)=251 nm. R_(f) 0.67 (MeOH/CH₂Cl, 20/80, v/v).

Example 11 Preparation of B242, the Hydroxy-tBuSATEN-(4-trifluoromethyl)benzylphosphoramidate Derivative of2′-C-methylguanosine

Synthetic Scheme:

2′-C-Methylguanosin-5′-yl-N-(4-trifluoromethyl)-benzylaminyl-O-(triphenylmethyloxy-tert-butyl-S-acyl-2-thioethyl)phosphate(D1)

To a solution of compound B1 [See, Compound 7, Example 3, Procedure A](355 mg, 0.465 mmol) in anhydrous carbon tetrachloride (4.65 mL)4-trifluoromethylbenzylamine (331 μL, 2.324 mmol) was added. Thereaction mixture was stirred at room temperature for 1 h 30 andconcentrated under reduced pressure (bath temperature not exceeding 30°C.). The resulting residue was subjected to silica gel chromatography,eluting with a gradient 0-10% methanol in dichloromethane, to give D1 asa white solid. (420 mg, 96%). Compound D1: ¹H NMR (400 MHz, DMSO-d₆) δ7.77-7.20 (m, 20H, 3C₆H₅, C₆H₄CF₃ and H-8), 6.57 (ls, 2H, NH₂),5.84-5.75 (m, 2H, H-1′ and PNH), 5.50 (m, 1H, OH-3′), 4.26-3.86 (m, 8H,H-3′, H-4′, H-5′, CH₂O and CH₂N), 3.10 (t, 2H, CH₂S, J_(CH2S-CH2O)=4.0Hz), 3.03 (m, 2H, CH₂OTr), 1.11 (s, 6H, 2CH₃), 0.82 (s, 3H, CH₃). ¹³CNMR (100 MHz, DMSO-d₆): δ 204.0 (C═O), 157.2 (C-4), 154.2 (C-2), 151.3(C-6), 145.8-143.9 (4C, 3C₆H₅ and C₆H₄CF₃), 135.6 (C-8), 129.0-120.0(20C, 3C₆H₅ and C₆H₄CF₃), 117.0 (C-5), 91.0 (C-1′), 86.1 (C(C₆H₅)), 80.7(C-3′), 78.7 (C-2′), 73.3 (C-4′), 70.0 (CH₂OTr), 65.9 (CH₂O), 64.4(C-5′), 50.8 (C(CH₃)₂CH₂OTr), 44.2 (CH₂N), 28.8 (CH₂S), 22.7 (2C, 2CH₃),20.4 (CH₃). ³¹P NMR (162 MHz, DMSO-d₆): δ 9.80 (s), 9.64 (s). ¹⁹F NMR(376 MHz, DMSO-d₆): δ 60.8 (s). LR LC/MS (M+H⁺) 937.3 (M−H⁻) 935.4 (5.47min). UV: λ_(max)=254 nm. R_(f) 61 (MeOH/CH₂Cl, 15/85, v/v).

O-(Hydroxy-tert-butyl-S-acyl-2-thioethyl)-2′-C-methylguanosin-5′-yl-N-(4-trifluoromethyl)-benzylaminylphosphate B242 (Compound D2)

Compound D1 (400 mg, 0.427 mmol) was dissolved in dichloromethane (1.6mL) and treated with TFA (160 μL). The mixture was stirred at roomtemperature for 15 min, then diluted with ethanol, evaporated to dryness(bath temperature not exceeding 30° C.) and coevaporated with toluene.The resulting residue was subjected to silica gel chromatography,eluting with a gradient 0-15% methanol in dichloromethane and thenpurified by reverse phase (C 18) silica gel column chromatographyeluting with a gradient 0-100% acetonitrile in water and lyophilisedfrom a mixture of water/dioxan to give compound B2742 (Compound D2) (90mg, 30%, white lyophilised powder). B242 (Compound D2): ¹H NMR (400 MHz,DMSO-d₆) δ 10.54 (ls, 1H, NH), 7.75 (s, 1H, H-8), 7.75-7.52 (m, 4H,C₆H₄CF₃), 6.50 (sl, 2H, NH₂), 5.82-5.74 (m, 2H, H-1′ and PNH), 5.40 (m,1H, OH-3′), 5.17 (s, 1H, OH-2′), 4.92 (t, 1H, OH, J_(OH-CH2)=4.0 Hz),4.26-3.84 (m, 8H, H-3′, H-4′, H-5′, CH₂O and CH₂N), 3.41 (d, 2H, CH₂OH,J_(CH2-OH)=4.0 Hz), 3.03 (t, 2H, CH₂S, J_(CH2S-CH2O)=8.0 Hz), 1.07 (s,6H, 2CH₃), 0.82 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ 204.4 (C═O),157.2 (C-4), 154.1 (C-2), 151.2 (C-6), 145.9 (C₆H₄CF₃), 135.8 (C-8),128.3-125.4 (6C, C₆H₄CF₃), 117.0 (C-5), 90.9 (C-1′), 80.5 (C-3′), 78.7(C-2′), 73.2 (C-4′), 68.8 (CH₂OH), 66.0 (CH₂O), 64.4 (C-5′), 52.2(C(CH₃)₂CH₂OH), 44.3 (CH₂N), 28.7 (CH₂S), 22.3 (2C, 2CH₃), 20.4 (CH₃).³¹P NMR (162 MHz, DMSO-d₆): δ 9.62 (s), 9.77 (s). ¹⁹F NMR (376 MHz,DMSO-d₆): δ−60.8 (s). LR LC/MS (M+H⁺) 695.2 (M−H⁻) 693.4 (4.25 min).HRFAB-MS C₂₆H₃₅O₉N₆F₃PS (M+H⁺) calculated 695.1876. found 695.1874. UV:λ_(max)=253 nm. R_(f) 0.43 (MeOH/CH₂Cl, 20/80, v/v).

Example 12 Preparation of B503, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of9-[(2R)-2-deoxy-2-fluoro-2-C-ethynyl-β-D-erythro-furanosyl]-guanine

Synthetic Scheme:

{9-[(2R)-2-Deoxy-2-fluoro-2-C-ethynyl-β-D-erythro-furanosyl]-guanin}-5′-yl-O-(triphenylmethyloxy-tert-butyl-5-acyl-2-thioethyl)H-phosphonate (G1)

Compound B5 [Unpublished results] (100 mg, 0.32 mmol) and compound F3[See Compound 5 of Example 2] (246 mg, 0.42 mmol) were coevaporatedtogether with anhydrous pyridine and dissolved in this solvent (4.8 mL).Pivaloyl chloride (80 μL, 0.64 mmol) was added dropwise at −15° C. andthe solution was stirred at the same temperature for 2 h. The reactionmixture was diluted with dichloromethane and neutralised with an aqueoussolution of NH₄Cl 0.5M. The mixture was partitioned betweendichloromethane and aqueous NH₄Cl 0.5M, the organic phases werecombined, dried over Na₂SO₄ evaporated under reduced pressure (bathtemperature not exceeding 30° C.) and coevaporated twice with toluene.The crude mixture was purified by flash column chromatography elutingwith a gradient 0-10% methanol in dichloromethane+0.2% acetic acid) toafford the desired product G1 as a colorless oil (68 mg, 28%). CompoundG1: ¹H NMR (400 MHz, DMSO-d₆): δ 10.72 (ls, 1H, NH), 7.83 (s, 1H, H-8),7.35-7.11 (m 15H, 3C₆H₅), 6.59 (m, 2H, NH₂), 6.36 (d, 1H, OH-3′,J_(OH-3′)=7.6 Hz), 6.14 (d, 1H, H-1′, J_(1′-F)=18.0 Hz), 4.65 (m, 1H,H-3′), 4.40-4.33 (m, 2H, H-5′), 4.10-4.01 (m, 3H, H-4′ and CH₂O), 3.93(d, 1H, CCH, ⁴J_(H-F)=5.6 Hz), 3.15-3.12 (m, 2H, CH₂S), 3.04 (s, 2H,CH₂OTr). ³¹P NMR (162 MHz, DMSO-d₆): δ 9.50 (s), 9.22 (s). ¹⁹F NMR (376MHz, DMSO-d₆): 8-156.5 (m). LR LC/MS (B) (M+Na⁺) 798.2 (M−H⁻) 774.2(4.93 min). UV: λ_(max)=254 nm. R_(f) 0.48 (MeOH/CH₂Cl, 15/85, v/v).

N-Benzylaminyl-{9-[(2R)-2-deoxy-2-fluoro-2-C-ethynyl-β-D-erythro-furanosyl]-guanin}-5′-yl-O-(triphenylmethyloxy-tert-butyl-S-acyl-2-thioethyl)phosphate

To a solution of compound G1 (68 mg, 0.088 mmol) in anhydrous carbontetrachloride (880 μL), benzylamine (48 μL, 0.44 mmol) was addeddropwise. The reaction mixture was stirred at room temperature for 2 hand evaporated to dryness (bath temperature not exceeding 30° C.). Thecrude mixture was filtered on a silica gel plug, eluting with a gradient0-10% methanol in dichloromethane to give compound G2 as a white solid(80 mg, quantitative yield). Compound G2: ³¹P NMR (162 MHz, DMSO-d₆): δ9.95 (s) 9.80 (s). ¹⁹F NMR (376 MHz, DMSO-d₆): δ−157.5 (m). LR LC/MS (B)(M+H⁺) 881.3 (M−H⁻) 879.4 (5.18 min). UV: λ_(max)=254 nm. R_(f) 0.31(MeOH/CH₂Cl, 15/85, v/v).

N-Benzylaminyl-{9-[(2R)-2-deoxy-2-fluoro-2-C-ethynyl-β-D-erythro-furanosyl]-guanin}-5′-yl-O-(hydroxy-tert-butyl-S-acyl-2-thioethyl)phosphateB503 (Compound G3)

Compound G2 (80 mg, 0.09 mmol) was dissolved in dichloromethane (320 μL)and treated with TFA (32 μl). The mixture was stirred at roomtemperature for 10 min, filtered through a solid phase extraction columneluting with a gradient 0-30% methanol in dichloromethane, then purifiedby reverse phase (C18) silica gel column chromatography eluting with agradient 0-100% acetonitrile in water and lyophilised from a mixture ofwater/dioxan to give compound B503 (Compound (15 mg, 26%, whitelyophilised powder). B503 (Compound C3): ¹H NMR (400 MHz, DMSO-d₆): δ10.61 (ls, 1H, NH), 7.83 (s, 1H, H-8), 7.30-7.18 (m, 5H, C₆H₅), 6.60(ls, 2H, NH₂), 6.32 (m, 1H, OH-3′), 6.11 and 6.12 (2 d, 2×1H, 2H-1′,J_(1′-F)=18.0 Hz), 5.68 (m, 1H, PNH), 4.93 (t, 1H, OH, J_(OH-CH2)=5.5Hz), 4.61 (m, 1H, H-3′), 4.26-4.18 (m, 2H, H-5′), 4.08 (m, 1H, H-4′),3.98-3.82 (m, 5H, CH₂O, CH₂N and CCH), 3.42 (d, 2H, CH₂OH,J_(CH2-OH)=5.0 Hz), 3.01 (m, 2H, CH₂S), 1.09 (s, 6H, 2CH₃). ³¹P NMR (162MHz, DMSO-d₆): δ 9.92 (s), 9.79 (s). ¹⁹F NMR (376 MHz, DMSO-d₆): δ−156.8(m). LR LC/MS (B) (M+H⁺) 639.2 (M−H) 637.3 (3.85 min). HRFAB-MSC₂₆H₃₃O₈N₆FPS (M+H⁺) calculated 639.1802. found 639.1816. UV:λ_(max)=253 nm. R_(f) 0.46 (MeOH/CH₂Cl, 20/80, v/v).

The starting nucleoside was synthesized as follows:

Synthesis of9-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-furanosyl]-guanine(D961, starting nucleoside of EXAMPLE 12), and synthesis of itstriphosphate derivative B427)

Synthetic Scheme:

9-[3,5-O-(1,3-Diyl-1,1,3,3-tetraisopropyldisiloxane)-ribo-furanosyl]-N²-isobutyryl-guanine(B1): Hirao, I.; Ishikawa, M.; Miura, K. Chem. Lett. 1986, 11,1929-1932.

9-[3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethylsilylethynyl-β-D-arabino-furanosyl]-N²-isobutyryl-guanine(B2): To a suspension of CrO₃ (11.07 g, 110.76 mmol) in dichloromethane(220 mL) at 0° C., acetic anhydride (10.4 mL, 110.76 mmol) and anhydrouspyridine (17.82 mL, 221.52 mmol) were added. Compound B1 (22 g, 36.92mmol) in solution in dichloromethane (110 mL) was added dropwise. Thecooling bath was removed and the resulting solution stirred at roomtemperature for 5 h. The reactionmixture was poured into cold ethylacetate, filtered through a silica and celite gel plug, concentrated todryness and coevaporated twice with toluene. The residue obtained wasdissolved in dichloromethane and stirred with an excess of MgSO₄overnight, filtered and evaporated to get the ketone. Thetrimethylsilylacetylene (12.5 mL, 88.60 mmol) was dissolved in anhydrousTHF (98 mL) under argon. Butyllithium (55.4 mL, 1.6 M in hexanes) wasadded dropwise at −78° C. The reaction mixture was stirred for 30 min at−78° C. and then allowed to warm up to −55° C. The ketone in solution inTHF (49 mL) was added dropwise at −78° C. The reaction mixture wasstirred for 1 h at −78° C. and then allowed to warm up to −30° C. andstirred for 3 h. The reaction was quenched by careful addition ofaqueous saturated NH₄Cl (72 mL) at −78° C. After warming to roomtemperature, the mixture was diluted with ethyl acetate, washed twicewith saturated brine, dried (Na₂SO₄) and concentrated to dryness. Thecrude material was purified using column chromatography eluting with1.5% MeOH in dichloromethane to give compound B2 (8.59 g, 34%, 2 steps)as a pale yellow foam. Compound B2: NMR ¹H (250 MHz, DMSO-d₆): δ 12.10(ls, 1H, NH), 11.69 (ls, 1H, NH), 7.91 (s, 1H, H-8), 6.69 (s, 1H, OH),5.94 (s, 1H, H-1′), 4.29 (d, 1H, H-3′, J_(3′-4)=5.5 Hz), 3.85-3.95 (m,3H, H-4′, H-5′ and H-5″), 2.46 (m, 1H, CH(CH₃)₂), 0.90-1.08 (m, 30H, iPrand CH(CH₃)₃), 0.00 (s, 9H, Si(CH₃)₂). LC/MS (A): (M+H⁺) 692.4 (24.96min). UV: λ_(max1)=254 nm, λ_(max2)=281 nm. R_(f) 0.34 (MeOH/CH₂Cl,15/85, v/v).

9-[(2R)-2-Deoxy-2-fluoro-3,5-O(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethyl-silylethynyl-β-D-erythro-furanosyl]-N²-isobutyryl-guanine(B)

Compound B2 (2.00 g, 2.89 mmol) was dissolved in dried DCM (60 mL) underargon and pyridine (1.45 mL, 18.06 mmol) was added. The reaction mixturewas cooled to −20° C. and DAST (4.11 mL, 31.35 mmol) was added dropwise.The cooling bath was removed after completion of the addition. Stirringwas continued for 1 h 15 and the mixture was dissolved with ethylacetate and poured into saturated NaHCO₃ and stirred for 5 min. Theorganic layer was washed with saturated brine, dried (Na₂SO₄),concentrated, and purified by silica gel chromatography eluting withethyl acetate in DCM (2%) to give the desired compound B3 (1.41 g, 70%)as a yellow oil. Compound B3: NMR ¹H (250 MHz, DMSO-d₆): δ 12.22 (s, 1H,NH), 8.09 (s, 1H, H-8), 6.21 (d, 1H, H-1′, J_(1′-F)=15.6 Hz), 4.54 (dd,1H, H-3′, J_(3′-F)=23.6 Hz, J_(3′-4′)=9.8 Hz), 4.33 (m, 1H, H-5′,²J_(5′-5″)=13.1 Hz), 4.16 (m, 1H, H-5″), 2.81 (m, 1H, CH(CH₃)₂),1.13-1.03 (m, 34H, iPr and CH(CH₃)₂), 0.08 (s, 9H, Si(CH₃)₃,³J_(H-H)=6.9 Hz). NMR ¹⁹F (235 MHz, DMSO-d₆): δ−160.26 (dd,J_(F-1′)=16.1 Hz, J_(F-3′)=23.3 Hz). LC/MS (A): (M+H⁺) 694.7 (24.02min). LRFAB-MS (GT): 694 (M+H)⁺, 692 (M−H)⁻. UV: λ_(max)=256 nm. R_(f)0.46 (MeOH/CH₂Cl, 05/95, v/v).

9-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-furanosyl]-N²-isobutyryl-guanine(B4)

Compound B3 (1.31 g, 1.89 mmol) was dissolved in methanol (13.8 mL) andammonium fluoride (908.9 mg, 24.54 mmol) was added. The resultingsolution was stirred at reflux for 1 h and evaporated to dryness. Thecrude material was purified on silica gel chromatography eluting with astepwise gradient 6-10% of methanol in dichloromethane to yield compoundB4 (344 mg, 48%) as a pale yellow oil. Compound B4: NMR ¹H (400 MHz,DMSO-d₆): δ12.18 (ls, 1H, NH), 11.77 (ls, 1H, NH), 8.34 (s, 1H, H-8),6.29 (d, 1H, OH-3′, J_(OH-3′)=7.5 Hz), 6.20 (d, 1H, H-1′, J_(1′-F)=16.2Hz), 5.39 (t, 1H, OH-5′, J_(OH-5′)=5.1 Hz), 4.52 (dt, 1H, H-3′,J_(3′-F)=22.9 Hz), 3.98 (m, 1H, H-4′), 3.90-3.85 (m, 2H, H-5′ andethynyl), 3.72 (m, 1H, H-5″), 2.52 (m, 1H, CH(CH₃)₂), 1.14 (d, 6H,CH(CH₃)₂, ³J_(H-H)=6.9 Hz). NMR ¹³C (100 MHz, DMSO-d₆: δ 180.7 (C-6),155.3 (C-2), 148.9 (C-4), 137.3 (C-8), 120.4 (C-5), 95.8 (d, C-2′,¹J_(2′-F)=182.1 Hz), 87.7 (d, C-1′, ²J_(1′-F)=39.2 Hz), 83.4 (d, CCH,³J_(C-F)=9.1 Hz), 82.6 (C-4′), 75.9 (d, CCH, ²J_(C-F)=31.2 Hz), 72.9 (d,C-3′, ²J_(3′-F)=19.1 Hz), 59.3 (C-5′). NMR ¹⁹F (235 MHz, DMSO-d) δ−158.9(m). LC/MS (A): (M+H)⁺) 380.3 (8.34 min). UV: λ_(max1)=260 nm,λ_(max2)=277 nm. R_(f) 0.40 (MeOH/CH₂Cl, 15/85, v/v).

9-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-furanosyl]-guanine D961(Compound B5)

Compound B4 (0.78 g, 1.33 mmol) was dissolved in saturated methanolicammonia (62 mL) and stirred at room temperature for 20 h. The reactionmixture was then evaporated to dryness under reduced pressure. Theresidue was dissolved in water and washed twice with ethyl acetate. Theaqueous layer was evaporated and purified on reverse phase columnchromatography (C 18) eluting with a gradient 2-15% of acetonitrile inwater. The residue obtained was then dissolved in hot ethyl acetate,filtered and dried to give D961 (Compound B5) (134 mg, 33%) as a yellowsolid. NMR ¹H (400 MHz, DMSO-d₆): δ 10.70 (ls, 1H, NH), 7.98 (s, 1H,H-8), 6.60 (ls, 2H, NH₂), 6.21 (d, 1H, OH-3′, J_(OH-3′)=7.6 Hz), 5.83(d, 1H, H-1′, J_(1′-F)=16.9 Hz), 5.29 (t, 1H, OH-5′, J_(OH-5′)=5.2 Hz),4.50 (td, 1H, H-3′, J_(3′-F)=22.8 Hz, J_(3′-4)=9.2 Hz), 3.93-3.81 (m,3H, H-4′, H-5′ and ethynyl), 3.70 (m, 1H, H-5″). NMR ¹³C (100 MHzDMSO-d₆): δ 157.2 (C-6), 154.3 (C-2), 151.05 (C-4), 135.1 (C-8), 116.7(C-5), 96.4 (d, C-2′, ¹J_(C-F)=182.1 Hz), 87.4 (d, C-1′, ²J_(C-F)=39.2Hz), 83.1 (d, CCH, J_(C-F)=9.1 Hz), 82.4 (C-4′), 76.2 (d, CCH,²J_(C-F)=31.2 Hz), 73.2 (d, C-3′, ²J_(C-F)=20.1 Hz), 59.5 (C-5′). NMR¹⁹F (235 MHz, DMSO-d₆): δ −158.5 (m). LC/MS (A): (M+H⁺) 310.1 (5.55min). LRFAB-MS (GT): 619 (2M+H)⁺, 310 (M+H)⁺, 152 (B+H)⁺, 617 (2M−H)⁺,308 (M−H)⁻. UV: λ_(max)=254 nm.

Synthetic Scheme:

Standard procedure for preparation of nucleoside 5′-triphosphate:(Ludwig, J. Acta Biochim. Biophys. Acad. Sci. Hung. 1981, 16, 131-133.)

To a solution of nucleoside (0.286 mmol) in triethylphosphate (750 μl),phosphoryle chloride (75 μL, 0.807 mmol) was added at 0° C. Thisreaction mixture A was stirred overnight at 5° C. Tributylammoniumpyrophosphate (PPi/Bu₃N 1/1.5, 1 g, 2.19 mmol) was dissolved inanhydrous DMF (2 mL). Tributylamine (420 μL, 1.76 mmol) was added to thePPi and the resulting mixture was stirred for 15 min at 0° C. 2.4 mL ofthis solution were added to the reaction mixture A. The reaction mixturewas stirred at 0° C. for 1 min. The reaction was carefully quenched withTEAB 1M (pH=7.5, 10 mL), stirred 20 min at 0° C., then diluted withwater and ethyl acetate. The aqueous phase was concentrated underreduced pressure. The crude material was subjected to DEAE-Sephadexchromatography eluting with a gradient 10⁻³−1 M of TEAB). The desiredfractions were combined, concentrated under reduced pressure andcoevaporated with a mixture of water/methanol, and finally coevaporatedwith water. The resulting residue was purified on semipreparative HPLC.Fractions containing the expected product were concentrated underreduced pressure, coevaporated whit a mixture of water/methanol andlyophilised from water. The triethylammonium salt triphosphate waseluted three times with water on a Dowex Na⁺ resin column to yield afterlyophilisation from water to the sodium salt.

9-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-furanosyl]-guanine5′-triphosphate sodium salt (B427): ¹H NMR 400 MHz, D₂O: δ 7.97 (s, 1H,H-8), 6.19 (d, 1H, H-1′, ³J_(1′-F)=16.0 Hz), 4.70 (m, 1H under H₂O,H-3′), 4.39 (m, 1H, H-5′), 4.29-4.22 (m, 2H, H-4′ and H-5″), 2.98 (d,1H, ethynyl, ⁴J_(H-F)=5.0 Hz). ³¹P NMR (162 MHz, D₂O): −10.50 (d, 1P,P_(γ), J_(Pγ-Pβ)=19.4 Hz), −11.03 (d, 1P, P_(α), J_(Pα-Pβ)=19.4 Hz),−22.38 (t, 1P, P_(β), J_(Pβ-Pγ)=J_(Pβ-Pα)=19.4 Hz). NMR ¹⁹F (376 MHzDMSO-d₆): δ−159.1 (m). LRFAB-MS (GT): 638 (M+Na)⁺, 616 (M+H)⁺, 594(M−Na+2H)⁺, 572 (M−2Na+3H)⁺, 550 (M−3Na+4H)⁺, 592 (M−Na)⁻, 570(M−2Na+H)⁻, 548 (M−3Na+2H)⁻.

Example 13 Preparation of B306, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of2′-C-methyl-5-aza-7-deaza-guanosine

Synthetic Scheme:

The pronucleotide B306 (25 mg, 6% overall yield) has been synthesizedfrom its parent nucleoside 2′-C-methyl-5-aza-7-deaza-guanosine (200 mg,0.67 mmol) following a similar procedure than the one described for thesynthesis of the pronucleotide prepared in the Example 4 and isolated asa white lyophilized powder. ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 0.90-0.91(d, J=2.56 Hz, 3H), 1.09 (d, J=4.26 Hz, 6H), 3.07-3.10 (t, J=6.66 Hz,2H), 3.42 (d, J=5.64 Hz, 2H), 3.86-3.99 (m, 6H), 4.10-4.15 (m, 1H),4.15-4.20 (m, 1H), 4.90-4.93 (t, J=5.64 Hz, 1H), 5.28 (s, 1H), 5.46-5.50(m, 1H) 5.62-5.69 (m, 1H), 5.80 (s, 1H), 7.00 (s, 2H), 7.18-7.21 (m,2H), 7.26-7.33 (m, 5H); ³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm) 9.80-9.95(2s); Scan ES+ 627 (M+H)⁺, λ_(max)=261.7 nm; HPLC (0-100% ACN over aperiod of 8 min) t_(R)=3.18 min λ_(max)=258.4 nm.

Example 14 Preparation of B389, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of 2′-C-methyl-7-deaza-guanosine

Synthetic Scheme:

The pronucleotide B389 (80 mg, 21% overall yield) has been synthesizedfrom its parent nucleoside 2′-C-methyl-7-deaza-guanosine (200 mg, 0.67mmol) following a similar procedure than the one described for thesynthesis of the pronucleotide prepared in the Example 4 and isolated asa white lyophilized powder. ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 0.74 (s,3H), 1.09 (s, 6H), 3.0 (t, J=6.10 Hz, 2H), 3.42 (d, J=5.49 Hz, 2H),3.8-4.0 (2m, 6H), 4.04-4.11 (m, 1H), 4.24-4.17 (m, 1H), 4.90-4.93 (t,J=5.36 Hz 1H), 4.96-4.98 (d, J=4.76 Hz, 1H), 5.31-5.36 (m, 1H),5.57-5.67 (m, 1H), 5.93 (s, 1H), 6.21-6.26 (m, 3H), 6.76 (d, J=22 Hz,1H), 7.19-7.23 (m, 1H), 7.27-7.32 (m, 4H), 10.34 (brs, 1H); ³¹P NMR(DMSO-d₆, 162 MHz) δ (ppm) 9.77 and 9.90 (2s); Scan ES⁺ 626 (M+H)⁺,λ_(max)=258.7 nm; HPLC (0-100% ACN over a period of 8 min) t_(R)=3.84min λ_(max)=259.6 nm.

Example 15 Preparation of B288, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of 3′-C-methyluridine

Synthetic Scheme:

The pronucleotide B288 (34 mg, 3% overall yield) was synthesized fromits parent nucleoside 3′-C-methyl-uridine (513 mg, 1.99 mmol) followinga similar procedure than the one described for the synthesis of thepronucleotide prepared in the Example 4 and isolated as a whitelyophilized powder. ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 1.09 (s, 6H), 1.15(s, 3H), 3.00-3.05 (m, 2H), 3.30 (s, 1H), 3.42 (d, J=6.13 Hz, 2H),3.76-3.79 (m, 1H), 3.86-3.99 (m, 6H), 4.92-4.94 (t, J=5.40 Hz, 1H), 4.97(s, 1H), 5.47 (m, 1H), 5.59-5.62 (m, 1H), 5.67-5.78 (m, 1H), 5.83-5.87(m, 1H), 7.20-7.24 (m, 1H), 7.30 (m, 4H), 7.66-7.71 (m, 1H), 11.32 (brs,1H); ³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm) 9.66 and 9.95 (2s); Scan ES⁺ 588(M+H)⁺, λ_(max)=261.7 nm.

Example 16 Preparation of B350, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of 3′-C-methylguanosine

Synthetic Scheme:

3′-C-Methylguanosin-5′-yl-O-(triphenylmethyloxy-tert-butyl-S-acyl-2-thioethyl)H-phos-phonate (E1)

3′-C-Methylguanosine (NM76) (233.7 mg, 0.79 mmol) and compound A3 [SeeCompound 5 of Example 2] (504.9 mg, 0.87 mmol) were coevaporatedtogether with anhydrous pyridine and dissolved in this solvent (11.8mL). Pivaloyl chloride (193.7 μL, 1.57 mmol) was added dropwise at −15°C. and the solution was stirred at the same temperature for 2 h. Thereaction mixture was diluted with dichloromethane and neutralized withan aqueous solution of NH₄Cl 0.5M. The mixture was partitioned betweendichloromethane and aqueous NH₄Cl 0.5M, the organic phases werecombined, dried over Na₂SO₄ evaporated under reduced pressure (bathtemperature not exceeding 30° C.) and coevaporated twice with toluene.The crude mixture was filtered on a silica gel plug eluting with agradient 0-10% methanol in dichloromethane+0.2% acetic acid) to affordthe desired product E1 (250 mg, 42%). Compound E1: ³¹P NMR (162 MHz,DMSO-d₆): δ 9.93 (s), 9.13 (s). LR LC/MS (M+H⁺) 521.1 (5.88 min). UV:λ_(max)=262 nm. R_(f) 0.21 (MeOH/CH₂Cl, 15/85, v/v).

N-Benzylaminyl-3′-C-methylguanosin-5′-yl-O-(triphenylmethyloxy-tert-butyl-S-acyl-2-thioethyl)phosphate(E1)

To a solution of compound E1 (250 mg, 0.33 mmol) in anhydrous carbontetrachloride (3.3 mL), benzylamine (178 μL, 1.637 mmol) was addeddropwise. The reaction mixture was stirred at room temperature for 1 h30 and evaporated to dryness (bath temperature not exceeding 30° C.).The crude mixture was filtered on a silica gel plug eluting with agradient 0-30% methanol in dichloromethane) to give compound E2 as awhite solid (290 mg, quantitative yield). Compound E2: ³¹P NMR (162 MHz,DMSO-d₆) δ 9.91 (s), 9.74 (s). LR LC/MS (M+H⁺) 869.3 (M−H⁻) 867.7 (5.20min). UV: λ_(max)=253 nm. R_(f) 0.13 (MeOH/CH₂Cl, 10/90, v/v).

N-Benzylaminyl-O-(hydroxyl-tert-butyl-S-acyl-2-thioethyl)-3′-C-methylguanosin-5′-ylphosphate B350 (Compound E3)

Compound E2 (290 mg, 0.33 mmol) was dissolved in dichloromethane (1.16mL) and treated with TFA (113 μL). The mixture was stirred at roomtemperature for 10 min, then diluted with ethanol, evaporated to dryness(bath temperature not exceeding 30° C.) and coevaporated with toluene.The resulting residue was subjected to silica gel chromatography,eluting with a gradient 0-30% methanol in dichloromethane, then purifiedby reverse phase (C 18) silica gel column chromatography eluting with agradient 0-100% acetonitrile in water and lyophilised from a mixture ofwater/dioxan to give B350 (Compound E3) (15 mg, 7%, white lyophilisedpowder). B350 (Compound E3): ¹H NMR (400 MHz, DMSO-d₆) δ 10.60 (m, 1H,NH), 7.90 (s, 1H, H-8), 7.30-7.19 (m, 5H, C₆H₅), 6.47 (ls, 2H, NH₂),5.72-5.59 (m, 2H, H-1′ and PNH), 5.51 (d, 1H, OH-2′, J_(OH2′-1′)=8.0Hz), 4.94-4.92 (2H, OH-3′ and OH), 4.28 (m, 1H, H-2′), 4.01-3.83 (m, 7H,H-4′, H-5′, CH₂O and CH₂N), 3.41 (m, 2H, CH₂OH), 3.02 (t, 2H, CH₂S,J_(CH2S-CH2O)=6.0 Hz), 1.20 (s, 3H, CH₃), 1.09 (s, 6H, 2CH₃). ³¹P NMR(162 MHz, DMSO-d₆) δ 9.86 (s), 9.72 (s). LR LC/MS (M+H⁺) 627.2 (M−H⁻)625.5 (3.87 min). HRFAB-MS C₂₅H₃₆O₉N₆PS (M+H⁺) calculated 627.2002.found 627.2014. UV: λ_(max)=251 nm.

Example 17 Preparation of B305, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of1-[2-C-methyl-β-ribofuranosyl]-3-carboxamido-4-fluoro-pyrazole

Synthetic Scheme:

The pronucleotide B305 (28.3 mg, 8% overall yield) has been synthesizedfrom its parent nucleoside1-[2-C-methyl-β-ribofuranosyl]-pyrazolo-3-carboxamide-4-fluoro (180 mg,0.65 mmol) following a similar procedure than the one described for thesynthesis of the pronucleotide prepared in the Example 4 and isolated asa white lyophilized powder. ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 0.75 (s,3H), 1.08-1.09 (d, J=3.35 Hz, 6H), 2.98-3.02 (m, 2H), 3.40-3.42 (m, 2H),3.85-4.03 (m, 5H), 4.16-4.19 (m, 2H), 4.89-4.92 (m, 1H), 5.25-5.29 (m,2H), 5.55 (s, 1H), 5.56-5.64 (m, 1H), 7.19-7.22 (m, 1H), 7.26-7.50 (m,7H), 8.05 (d, J=4.32 Hz, 1H); ³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm) 9.75and 9.90 (2s); ¹⁹F NMR (d₆-DMSO, 235 MHz) δ (ppm) −170.70 (d, J=61.74Hz, 1F); Scan ES⁺ 605 (M+H)⁺, λ_(max)=233.7 nm; HPLC (0-100% ACN over aperiod of 10 min) t_(R)=4.56 min λ_(max)=235.2 nm.

Example 18 Preparation of B436, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of2′-C-methyl-7-deaza-7-fluoro-adenosine

Synthetic Scheme:

The pronucleotide B436 (30 mg, 9% overall yield) has been synthesizedfrom its parent nucleoside2′-C-methyl-7-deaza-6-NH-dimethoxytrityl-adenosine (320 mg, 0.53 mmol)following a similar procedure than the one described for the synthesisof the pronucleotide prepared in the Example 2 (Procedure A, Strategyb), and isolated as a white lyophilized powder. ¹H NMR (DMSO-d₆, 400MHz) δ (ppm) 0.66 (s, 3H), 1.02 (s, 6H), 2.95-2.98 (t, J=6.10 Hz, 2H),3.35 (d, J=5.49 Hz, 2H), 3.77-3.85 (m, 3H), 3.88-3.95 (m, 3H), 4.03-4.18(m, 2H), 4.83-4.86 (t, J=5.44 Hz, 1H), 5.14 (s, 1H), 5.21-5.25 (t, 7.40Hz, 1H), 5.55-5.66 (m, 1H), 6.14 (s, 1H), 6.9-7.3 (m, 8H), 8.01 (s, 1H);³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm) 9.77 and 9.89 (2s); ¹⁹F NMR (d₆-DMSO,235 MHz) δ (ppm) −166.85 (d, J=14.16 Hz, 1F); Scan ES⁺ 628 (M+H)⁺,λ_(max)=280.7 nm; HPLC (0-100% ACN over a period of 10 min) t_(R)=4.78min λ_(max)=280.8 nm.

Example 19 Preparation of B589, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of 4′-C-methyluridine

Synthetic Scheme:

Following the procedures described for Example 4, and starting from4′-C-methyluridine (A437) (200 mg, 0.77 mmol), intermediate P3 was firstproduced (62 mg, 11%. ³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm) 9.15, 9.56(2s); Scan ES⁺ 747 (M+Na)⁺, λ_(max)=259.7 nm), then compound P4 duringthe second step (28 mg, 39%. Scan ES⁺ 852 (M+Na)⁺), and finally thedesired prodrug B589 was obtained as a white powder after lyophilizationfrom dioxane (21 mg, 58%). ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 1.10 (s,6H), 1.13 (s, 3H), 3.03 (t, J=6.42 Hz, 2H), 3.15 (d, J=5.29 Hz, 1H),3.42 (d, J=5.67 Hz, 2H), 3.72-4.16 (m, 7H), 4.06-4.15 (m, 1H), 4.92 (t,J=5.29 Hz, 1H), 5.22 (d, J=5.29 Hz, 1H), 5.36-5.38 (2d, 1H), 5.57-5.60(2d, 1H), 5.64-5.70 (m, 1H), 5.78-5.80 (2d, 1H), 7.20-7.31 (m, 5H),7.60-7.64 (2d, 1H); ³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm) 9.58, 9.77 (2s);Scan ES⁺ 610 (M+Na)⁺, λ_(max)=260.7 nm.

Example 20 Preparation of B678, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of 4′-C-fluoromethylguanosine

Synthetic Scheme:

Following the procedures of the Procedure A described in Example 3, andstarting from 4′-C-fluoromethylguanosine (A402) (69.4 mg, 0.22 mmol),compound P1 (67.5 mg, 39%) was obtained as intermediate after the firststep. Scan ES⁻ 780 (M−H)⁻. Second step led to the formation ofintermediate P2 (57.5 mg; 76%). Compound P2 (26.3 mg, 0.03 mmol) wasdissolved in dichloromethane (1 ml) and treated with montmorillonite K10(150 mg) and stirred at room temperature for 1 h. The reaction mixturewas directly deposited on silica SPE tube and extracted with a gradient0-100% MeOH in dichloromethane to give after lyophilization fromdioxane/water B678 as a white powder (7.7 mg, 40%). ¹H NMR (DMSO-d₆, 400MHz) δ (ppm) 1.09 (2 ls, 6H), 3.03-3.05 (m, 2H), 3.42 (m, 2H), 3.87-4.00(m, 6H), 4.15-4.24 (m, 2H), 4.43-4.66 (m, 2H), 4.74 (m, 1H), 4.93 (m,1H), 5.52 (d, J=4.36 Hz, 1H), 5.58 (m, 1H), 5.70-5.73 (m, 1H), 5.75-5.77(d, J=8.05 Hz, 1H), 6.52 (ls, 2H), 7.24-7.35 (m, 5H), 7.92 (2s, 1H); ³¹PNMR (DMSO-d₆, 162 MHz) δ (ppm) 9.70, 9.83 (2s); ¹⁹F NMR (d₆-DMSO, 235MHz) δ (ppm) −235.92, −236.25 (2s); Scan ES⁺ 645 (M+H)⁺, λ_(max)=250.7nm HPLC (0-100% ACN over a period of 8 min) t_(R)=3.91 min λ_(max)=251.1nm.

Example 21 Preparation of B704, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of acyclovir

Synthetic Scheme:

9-(2-Hydroxy-ethoxymethyl)-guanin-5′-yl-O-(triphenylmethyloxy-tert-butyl-S-acyl-2-thioethyl)H-phosphonate(F1)

Acyclovir (200 mg, 0.89 mmol) and compound A3 [See Compound 5 of Example2] (674.2 mg, 1.15 mmol) were coevaporated together with anhydrouspyridine and dissolved in this solvent (13.3 mL). Pivaloyl chloride (162μL, 1.15 mmol) was added dropwise at −15° C. and the solution wasstirred at room temperature for 2 h. The reaction mixture was dilutedwith dichloromethane and neutralized with an aqueous solution of NH₄Cl0.5M. The mixture was partitioned between dichloromethane and aqueousNH₄Cl 0.5M, the organic phases were combined, dried over Na₂SO₄evaporated under reduced pressure (bath temperature not exceeding 30°C.) and coevaporated twice with toluene. The crude mixture was filteredon a silica gel plug eluting with a gradient 0-15% methanol indichloromethane+0.2% acetic acid) to afford the desired product F1 (602mg, 98%). Compound F1: LR LC/MS (M+H⁺) 691.9 (M−H⁻) 690.0 (4.82 min).UV: λ_(max)=254 nm.

N-Benzylaminyl-9-(2-hydroxy-ethoxymethyl)-guanin-5′-yl-O-(triphenylmethyloxy-tert-butyl-S-acyl-2-thioethyl)phosphate(F2)

To a solution of compound F1 (602 mg, 0.87 mmol) in anhydrous carbontetrachloride (8.7 mL), benzylamine (475 μL, 4.35 mmol) was addeddropwise. The reaction mixture was stirred at room temperature for 1 h30 and evaporated to dryness (bath temperature not exceeding 30° C.).The crude mixture was subjected to to silica gel chromatography, elutingwith a gradient 0-10% methanol in dichloromethane to give compound F2 asa white solid (550 mg, 79%). Compound F2: ¹H NMR (400 MHz, DMSO-d₆) Et7.77 (s, 1H, H-8), 7.58-7.17 (m, 20H, 4C₆H₅), 6.68 (ls, 2H, NH₂), 5.59(m, 1H, PNH), 5.32 (s, 2H, OCH₂N), 3.92-3.78 (m, 6H, CH₂SCH₂O, CH₂N,POCH₂CH₂O), 3.51 (t, 2H, POCH₂CH₂O, J_(CH2-CH2)=5.2 Hz), 3.16 (s, 2H,CH₂OTr), 3.00 (t, 2H, CH₂S, J_(CH2S-CH2O)=5.6 Hz), 1.12 (s, 6H, 2CH₃).¹³C NMR (100 MHz, DMSO-d₆): δ 204.0 (C═O), 157.2 (C-4), 154.6 (C-2),151.8 (C-6), 143.9 (4C, 4C₆H₅), 136.9 (C-8), 128.9-127.2 (20C, 4C₆H₅),117.0 (C-5), 86.3 (1C, C(C₆H₅)₃), 72.3 (OCH₂N), 70.0 (CH₂OTr), 68.2(POCH₂CH₂O), 64.8 (POCH₂CH₂O), 64.2 (CH₂SCH₂O), 50.8 (C(CH₃)₂), 44.7(CH₂N), 28.8 (CH₂S), 22.8 (2C, C(CH₃)₂). ³¹P NMR (162 MHz, DMSO-d₆) δ9.79 (s). LR LC/MS (M+H⁺) 797.2 (5.15 min). UV: λ_(max)=254 nm. R_(f)0.57 (MeOH/CH₂Cl, 15/85, v/v).

N-Benzylaminyl-9-(2-hydroxy-ethoxymethyl)-guanin-5′-yl-O-(hydroxy-tert-butyl-S-acyl-2-thioethyl)phosphateB704 (Compound F3)

Compound F2 (550 mg, 0.69 mmol) was dissolved in dichloromethane (2.2mL) and treated with TFA (220 μL). The mixture was stirred at roomtemperature for 15 min, filtered through a solid phase extraction columneluting with a gradient 0-15% methanol in dichloromethane, then purifiedby reverse phase (C 18) silica gel column chromatography eluting with agradient 0-100% acetonitrile in water and lyophilised from a mixture ofwater/dioxan to give B704 (Compound F3), (103 mg, 27%, white lyophilisedpowder). B704 (Compound F3): ¹H NMR (400 MHz, DMSO-d₆): δ 10.57 (ls, 1H,NH), 7.79 (s, 1H, H-8), 7.29-7.18 (m, 5H, C₆H₅), 6.49 (ls, 2H, NH₂),5.55 (m, 1H, PNH), 5.33 (s, 2H, OCH₂N), 4.92 (t, 1H, OH, J_(OH-CH2)=5.2Hz), 3.94-3.73 (m, 6H, CH₂SCH₂O, CH₂N, POCH₂CH₂O), 3.60 (t, 2H,POCH₂CH₂O, J_(CH2-CH2)=4.2 Hz), 3.42 (d, 2H, CH₂OH, J_(CH2-OH)=4.4 Hz),3.00 (t, 2H, CH₂S, J_(CH2S-CH2O)=6.4 Hz), 1.10 (s, 6H, 2CH₃). ¹³C NMR(100 MHz, DMSO-d₆): δ 204.4 (C═O), 157.2 (C-4), 154.4 (C-2), 151.9(C-6), 141.0 (1C, C₆H₅), 138.1 (C-8), 128.6-127.2 (5C, C₆H₅), 117.0(C-5), 72.3 (OCH₂N), 68.8 (CH₂OH), 68.2 (POCH₂CH₂O), 64.7 (POCH₂CH₂O),64.2 (CH₂SCH₂O), 52.2 (C(CH₃)₂), 44.7 (CH₂N), 28.7 (CH₂S), 22.3 (2C,C(CH₃)₂). ³¹P NMR (162 MHz, DMSO-d₄): δ 9.76 (s). LR LC/MS (M+H⁺) 555.9(M−H⁻) 553.9 (3.77 min). HRFAB-MS C₂₂H₃₂O₇N₆PS (M−1-H⁺) calculated555.1791. found 555.1795. UV: λ_(max)=250 nm.

Example 22 Preparation of B390, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of2′-C-methyl-2′,3′-di-O-acetyl-cytidine

Synthetic Scheme:

To a solution of pronucleotide 13 (See Example 2, Procedure A, Strategyb) (300 mg, 0.27 mmol) in anhydrous acetonitrile were successively addedtriethylamine (92 μl), acetic anhydride (2.2 eq, 54 μl) and4-dimethylaminopyridine (0.1 eq, 4 mg). The reaction mixture was stirredat room temperature for 2 h and triethylamine (92 μl), acetic anhydride(2.2 eq, 54 μl) and 4-dimethylaminopyridine (0.1 eq, 4 mg) were addedagain. After removal of the solvents under reduced pressure, the crudemixture was purified on silica gel column chromatography (eluant:stepwise gradient of methanol [0-5%] in methylene chloride) to give thefully protected pronucleotide (329 mg, quantitative yield). Thiscompound was finally treated with a mixture of trifluoroacetic acid (132μl) and methylene chloride (3.9 ml). After 1 h 30 stirring at roomtemperature trifluoroacetic acid (132 μl) were added again and themixture stirred 1 h more. The solvents were evaporated under reducedpressure and coevaporated with toluene. The crude mixture was purifiedon silica gel column chromatography (eluant: stepwise gradient ofmethanol [0-10%] in methylene chloride) to give B390 (36.4 mg, 21%)lyophilized as a white powder. ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 1.10(s, 6H), 1.33 (d, J=2.60 Hz, 3H), 2.05 (s, 6H), 3.01-3.04 (t, J=6.54 Hz,2H), 3.31 (d, J=5.45H, 2H), 3.85-3.90 (m, 2H), 3.94-3.99 (m, 2H),4.09-4.11 (m, 2H), 4.21-4.23 (m, 1H), 4.90-4.93 (t, J=5.71 Hz, 1H), 5.22(m, 1H), 5.67-5.73 (m, 2H), 6.20 (m, 1H), 7.21-7.27 (m, 7H), 7.54 (m,1H); ³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm) 9.69 and 9.86 (2s); Scan ES⁺ 671(M+H)⁺, λ_(max)=273.7 nm; HPLC (0-100% ACN over a period of 10 min)t_(R)=5.04 min λ_(max)=233.7 nm and 271.4 nm.

Example 23 Preparation of B302

Synthesis of Hydroxy-tBuSATE N-benzylphosphoramidate 2′,3′-cycliccarbonate Derivative of 2′-C-methylcytidine B302

The following strategy was used for the synthesis:

Protected phosphoramidate 13 (1.72 g, 1.52 mmol) was dissolved inanhydrous dichloromethane (17 ml) under argon. 1,1′-Carbonyldiimidazole(251 mg, 1.55 mmol) was added and the reaction mixture was stirred atroom temperature under argon for 1 h.

Analysis by TLC (8% MeOH in DCM) indicated incomplete conversion ofstarting material (Rf 0.35) to product (Rf 0.56). HPLC analysis (methodTest 20, 272 nm) confirmed the profile: 9% starting material (Rt 7.30min) and 91% product (Rt 7.97 min).

Further portions of CDI (final total 299 mg, 1.84 mmol) were added andthe reaction mixture was left to stir for an additional 24 h at roomtemperature after which time analysis by HPLC indicated 1.5% SM and97.5% P.

The reaction mixture was evaporated in vacuo to give an off-white foam(1.97 g). Purification by silica gel plug column, eluting with ethylacetate, and evaporation of the appropriate fractions gave the protectedcyclic carbonate 14 (C₆₅H₆₅N₄O₁₂PS 1157.27 gmol⁻¹) as a white foam (1.62g, 92% yield). TLC (8% MeOH in DCM): Rf 0.56; HPLC Test 20 AUC:99.5%@254 nm, Rt 7.97 min; m/z (ESI−): 1155.9 [M−H]⁻ 100%; m/z (ESI+):1157.5 [M+H]⁺ 100%, 1179.5 [M+Na]⁺ 20%.

Protected cyclic carbonate 14 (1.50 g, 1.30 mmol) was dissolved inanhydrous dichloromethane (15 ml) at room temperature under argon. Neattrifluoroacetic acid (1.77 g, 15.5 mmol) was added dropwise to thereaction mixture which was then stirred for 45 min at room temperature.Analysis by HPLC (method Test 20, 272 nm) indicated disappearance ofstarting material (Rt 7.97 min) and formation of product (Rt 3.80 min).

Anhydrous methanol (5 ml) was added to the reaction mixture and solvents(10 ml) were partially removed in vacuo at 25° C. Further methanol (7ml) was added to the mixture which was then evaporated to give an orangeresidue. Trituration with hexane/TBME 3:2 (12 ml) for 20 min yielded asticky residue plus an opaque supernatant which was decanted.Retrituration with hexane/TBME 3:2 (5 ml) for 1 h and removal of thesecond supernatant gave, after coevaporation with methanol (3 ml), apale foam (1.18 g).

The crude foam was purified by reverse phase chromatography (loaded in 1ml acetonitrile and eluted with 0%, 10%, 15%, 20%, 25%, 30% acetonitrilein water). Combination of the relevant fractions, evaporation of thesolvents at 25° C. and chasing with ethanol (1 ml) gave cyclic carbonate15, B302, as a white foamy solid (560 mg, 71% yield).

B302: C₂₅H₃₃N₄O₁₀PS 612.59 gmol⁻¹

HPLC AUC (Method Test 20): 99%@254 nm, Rt 3.83 min

m/z (ESI+): 613.1 [M+H]⁺ 100%; 1225.5 [2M+H]⁺ 100%; 453.1 [N+H]⁺ 95%

m/z (ESI−): 611.4 [M−H]⁻ 80%; 1223.9 [2M−H]⁻ 50%; 451.3 [N−H]⁻ 100%

v_(max) (KBr disc) (cm⁻¹): 3346.4, 3206.5 O—H, intermolecular H-bond;1815.3 C═O cyclic 5-ring carbonate; 1650.9 br C═O base, thioester

KF: 1.54% H₂O content

Specific Rotation: [α]_(D) ²⁰+9.289 (c 10.104 mg cm⁻³ in DMSO)

m.p.: 100-102° C. contracts and softens, 104-106° C. phase transition I,127-135° C. phase transition II to a sticky glass, 140-150° C. partialmelting to sticky residue, 200-210° C. decomposes to a brown stickymaterial

Elemental Analysis: Calculated: C, 49.02%; H, 5.43%; N, 9.15%.

Found: C, 49.30%; H, 5.26%; N, 9.30%—passed with 0.26% F present (fromTFA)

NMR: Assigned using ¹H, ¹³C, ³¹P), COSY, TOCSY, DEPT, HSQC and HMBC

H NMR δ_(H) (400 MHz, d6-DMSO): 1.11 (6H, s, (CH₃)₂C), 1.30 (3H, br-s,CH₃), 3.04 (2H, m, CH₂S), 3.44 (2H, d, J=4 Hz, CH₂OH), 3.87-3.92 (2H, m,CH₂O), 3.94-4.01 (2H, m, CH₂Ph), 4.15-4.25 (2H, m, H-5′, H-5″), 4.37(1H, br-s, H-4′), 4.95 (2H, br-s, H-3′, CH₂OH), 5.75-5.77 (2H, 2×d, J=7Hz, H-5, P—N—H), 6.07 (1H, br-s, H-1′), 7.22-7.25 (1H, m, Ar—H),7.29-7.33 (4H, m, 4×Ar—H), 7.39, 7.44 (2H, 2×br-s, NH₂), 7.62 (1H, br-d,J=7 Hz, H-6)

¹³C NMR δ_(C) (100 MHz, d6-DMSO): 17.72 (CH₃), 21.78 (C(CH₃)₂), 28.13,28.21 (CH₂S), 44.17 (PhCH₂), 51.62 (C(CH₃)₂), 63.84, 63.89 (CH₂O), 64.55(C-5′), 68.29 (CH₂OH), 94.23 (C-5), 126.70 (Ar—C_(para)), 127.08, 128.11(2×Ar—C_(meta), 2×Ar—C_(ortho)), 140.35, 140.38 (Ar—C_(ipso)), 152.73,154.45 (C-2, C-4), 165.69 (C-6), 203.87 (C═OS). C-1′, C-2′, C-3′, C-4′and C═O broadened into baseline and were not observed.

³¹P NMR δ_(p) (162 MHz, d6-DMSO): 9.80, 9.94 (1P, 2×s, ratio 1.15:1.00)

Example 24 Preparation of B234, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of 3′-O-L-valinyl-2′-C-methylcytidine

The following strategy was used for the synthesis:

Boc protected valine (6.72 g, 30.94 mmol) was dissolved in anhydrous DCM(50 ml) and 1,1′-carbonyldiimidazole (4.87 g, 30.01 mmol) was added atroom temperature under argon. Vigorous evolution of gas was observedinitially during the activation step and the mixture was stirred at roomtemperature for 30 min.

Protected phosphoramidate 13 (10.0 g, 8.84 mmol) was dissolved inanhydrous dichloromethane (50 ml) in a separate vessel under argon. Theactivated Boc-Val solution was added dropwise to the phosphoramidatesolution and the resulting mixture was heated to 40° C. under argon for24 h.

Analysis by TLC (8% MeOH in DCM) indicated complete conversion ofstarting material 13 (Rf 0.35) to product (Rf 0.50). HPLC analysis(method Test 20, 272 nm) confirmed the profile: starting material (Rt7.30 min) and product (Rt 9.46 min).

The reaction mixture was evaporated in vacuo to give an off-white foam.Purification by silica gel column chromatography, loaded from DCM,eluting with ethyl acetate/hexane 1:1 then 100% ethyl acetate, andevaporation of the appropriate fractions gave the protected valine ester16 (C₇₄H₈₄N₅O₁₄PS 1330.52 gmol⁻¹) as a white foam (10.3 g, 88% yield).TLC (ethyl acetate): Rf 0.24; HPLC Test 20 AUC: 97%@272 nm, Rt 9.46 min;m/z (ESI−): 1329.29 [M−H]⁻ 100%; m/z (ESI+): 1331.68 [M+H]⁺ 25%, 303.16[DMTr]⁺ 100%.

Protected valine ester 16 (3.0 g, 2.25 mmol) was dissolved in anhydrousdichloromethane (22.5 ml) at room temperature under argon. Neattrifluoroacetic acid (4.5 ml, 58.4 mmol) was added dropwise to thereaction mixture over 3 min which was then stirred for 1 h at roomtemperature. Analysis by HPLC (method Test 20, 272 nm) indicateddisappearance of starting material (Rt 9.46 min) and formation ofproduct (Rt 3.33 min) along with significant Boc intermediate (Rt 4.60min).

Additional neat trifluoroacetic acid (1.0 ml, 13.0 mmol) was addeddropwise to the reaction mixture which was then stirred for a further 1h at room temperature. Analysis by HPLC (method Test 20, 272 nm)indicated disappearance of Boc intermediate (Rt 4.60 min) and formationof product (Rt 3.33 min).

The reaction mixture was cooled to 5° C. and anhydrous methanol (50 ml)was added, stirring for 30 min. Solvents were removed in vacuo at 25° C.The residue was treated with TBME (50 ml×3) and triturated, decantingthe three TBME liquors.

The residual material was dissolved in anhydrous methanol (5 ml) andanhydrous DCM (10 ml) and solid sodium bicarbonate (5 g) was added,stirring for 30 min, to give pH 6. The clear liquid was passed through asyringe filter. The residual solid bicarbonate was washed with 25%methanol in DCM (anhydrous, 10 ml) and the solution was again filtered.The combined filtrates were concentrated in vacuo to give crude 17 (2.15g).

The crude material was purified by reverse phase chromatography (loadedin 15 ml water and 3 ml acetonitrile and eluted with 0%, 5%, 20%, 30%acetonitrile in water). Combination of the relevant fractions andevaporation of the solvents at 25° C. gave valine ester 17, B234, as awhite foamy solid (737 mg, 48% yield).

B234: C₂₉H₄₄N₅O₁₀PS 685.73 gmol⁻¹

HPLC AUC (Method Test 20): 99%@254 nm, Rt 3.33 min

m/z (ESI+): 686.3 [M+H]⁺ 100%; 1371.6 [2M+H]⁺ 20%; 526.1 [N+H]⁺ 20%

m/z (ESI−): 744.4 [M+OAc]⁻ 35%; 1369.8 [2M−H]⁻ 35%; 1430.2 [2M+OAc]⁻15%; 524.5 [N−H]⁻ 100%

v_(max) (KBr disc) (cm⁻¹): 3350.7, 3211.9 O—H, N—H, 1757.8 C═O ester;1673.9, 1652.0 C═O thioester, baseKF: 1.94% H₂O contentSpecific Rotation: [α]_(D) ²⁰+44.37° (c 10.033 mg cm⁻³ in DMSO)NMR: Assigned using ¹H, ¹³C, ³¹P, COSY, TOCSY, DEPT, HSQC and HMBC

H NMR δ_(H) (400 MHz, d6-DMSO): 0.96, 0.98 (2×314, 2×s, (CH₃)₂CH), 1.03(3H, br-s, CH₃), 1.11 (6H, s, (CH₃)₂C), 2.15 (1H, m, (CH₃)₂CH), 3.03(2H, m, CH₂S), 3.44 (2H, a-s, CH₂OH), 3.85 (1H, a-d, J=4.8 Hz, CHNH₂),3.85-3.92 (2H, m, CH₂O), 3.92-4.00 (2H, m, CH₂Ph), 4.06-4.11 (1H, br-m,H-5′), 4.17-4.20 (1H, br-m, H-5″), 4.27-4.29 (1H, br-m, H-4′), 5.08 (1H,br-s, H-3′), 5.73 (1H, a-t, J=7.3 Hz, H-5), 5.74-5.82 (1H, m, P—N—H),5.92 (1H, br-s, H-1′), 7.22-7.25 (1H, m, Ar—H), 7.28-7.32 (4H, m,4×Ar—H), 7.60, 7.63 (2×0.5H, 2×d, J=7.3 Hz, H-6). 2×O—H and 2×NH₂ notobserved.

¹³C NMR δ_(C) (100 MHz, d6-DMSO): 17.84, 17.96 (CH(CH₃)₂), 20.42, 20.48(CH₃), 21.78, (C(CH₃)₂), 28.09, 28.16 (CH₂S), 29.72 (CH(CH₃)₂), 44.16(PhCH₂), 51.62 (C(CH₃)₂), 57.84 (CHNH₂), 63.77, 63.81 (CH₂O, C-5′),68.26 (CH₂OH), 74.67 (C-3′), 77.28 (C-4′), 78.09 (C-2′), 91.32 (C-1′),94.22 (C-5), 126.71 (Ar—C_(para)), 127.04, 127.08, 128.11(2×Ar—C_(meta), 2×Ar—C_(ortho)), 140.23, 140.27, 140.32 (Ar—C_(ipso),C-6), 155.06 (C-2), 165.32 (C-4), 169.65, 169.72 (CO₂R), 203.84 (C═OS).C-1′, C-3′, C-4′ broadened into baseline but observable.

³¹P NMR δ_(P) (162 MHz, d6-DMSO): 9.63, 9.96 (1P, 2×s, ratio 1.02:1.00)

Example 25 Preparation of B183, the Hydroxy-tBuSATEN-benzylphosphoramidate Derivative of 2′-C-methyl-NH-4-acetyl-cytidine

Synthetic Scheme:

To a solution of B102 (See Example 2) (compound 10, 200 mg, 0.34 mmol)in anhydrous dimethylformamide (3.4 ml) was added dropwise aceticanhydride (1.1 eq, 34 μl). The reaction mixture was stirred at roomtemperature for 4 h and 10 μl of acetic anhydride were added again. Thereaction mixture was stirred overnight and the solvent evaporated underreduced pressure. The crude mixture was purified on silica gel columnchromatography (eluant: stepwise gradient of methanol [0-10%] inmethylene chloride) to give the desired acetylated pronucleotide B183(169 mg, 79%) isolated as a white lyophilized powder. ¹H NMR (DMSO-d₆,400 MHz) δ (ppm) 0.93 (s, 3H), 1.09 (s, 6H), 2.09 (s, 3H), 3.01-3.04 (t,J=6.54 Hz, 2H), 3.40-3.42 (d, J=5.10 Hz, 1H), 3.53-3.62 (m, 2H),3.83-3.91 (m, 1H), 3.94-4.01 (m, 4H), 4.10-4.15 (m, 1H), 4.20-4.25 (m,1H), 4.88-4.91 (t, J=5.20 Hz, 1H), 5.23 (s, 1H), 5.33-5.37 (t, J=7.19Hz, 1H), 5.67-5.78 (m, 1H), 5.93 (s, 1H), 7.18-7.21 (m, 1H), 7.27-7.32(m, 5H), 7.96 and 8.03 (2d, J=7.59 Hz, 1H), 10.87 (s, 1H); ³¹P NMR(DMSO-d₆, 162 MHz) δ (ppm) 9.74 and 9.98 (2s); Scan ES⁺ 629 (M+H)⁺,λ_(max)=300.7 nm; HPLC (0-100% ACN over a period of 8 min) t_(R)=4.89min λ_(max)=302.1 nm

Example 26 Preparation of B187, the Hydroxy-tBuSATEN-(2-(trifluoromethyl)benzyl)phosphosphoramidate Derivative of2′-C-methylcytidine

Synthetic Scheme:

To a solution of compound 8 (See Example 2, Procedure A, Strategy a)(1.4 g, 1.3 mmol) in anhydrous carbon tetrachloride (13 ml) was addeddropwise N-2-(trifluoromethyl)benzylamine (10 eq, 2.3 g). The reactionmixture was stirred at room temperature for 3 h and the solvent removedunder reduced pressure. The crude mixture was purified on silica gelcolumn chromatography (eluant: stepwise gradient [0-3%] of methanol inmethylene chloride) to afford the desired protected nucleoside as a foam(60%). This compound was converted into the phosphoramidate prodrug B187(245 mg, 35%) following experimental conditions described in the Example2 Strategy A and isolated as a white lyophilized powder. ¹H NMR(DMSO-d₆, 400 MHz) δ (ppm) 0.92 (s, 3H), 1.09 (s, 6H), 3.05 (t, J=6.45Hz, 2H), 3.29 (s, 1H), 3.41 (d, J=5.60 Hz, 2H), 3.91-3.93 (m, 3H),4.17-4.21 (m, 4H), 4.91 (t, J=5.59 Hz, 1H), 5.06 (d, J=4.25 Hz, 1H),5.23 (t, J=7.50 Hz, 1H), 5.65-5.67 (m, 1H), 5.76-5.83 (m, 1H), 5.91 (s,1H), 7.08 and 7.16 (2s, 2H), 7.45-7.79 (m, 5H); ³¹P NMR (DMSO-d₆, 162MHz) δ (ppm) 9.57-9.78 (2s, 1P); ¹⁹F NMR (d₆-DMSO, 235 MHz) δ (ppm)−60.79 (s, 3F); Scan ES⁺ 655 (M+H)⁺, λ_(max)=280.73 nm; HPLC (0-100% ACNover a period of 10 min) t_(R)=5.08 min λ_(max)=271.4 nm.

Example 27 Preparation of B399, the Hydroxy-tBuSATEN-(4-(trifluoromethyl)benzyl)phosphosphoramidate Derivative of2′-C-methylcytidine

Synthetic Scheme:

To a solution of compound 8 (See Example 2, Procedure A, Strategy a)(1.0 g, 0.94 mmol) in anhydrous carbon tetrachloride (10 ml) was addeddropwise N⁴-trifluoromethylbenzylamine (5 eq, 670 μl). The reactionmixture was stirred at room temperature for 3 h and the solvent removedunder reduced pressure. The crude mixture was purified on silica gelcolumn chromatography (eluant: stepwise gradient [0-5%] of methanol inmethylene chloride) to afford the desired protected nucleoside as a foam(84%). This compound was converted into the phosphoramidate prodrug B399(204 mg, 40%) following experimental conditions described in the Example2 Strategy A and isolated as a white lyophilized powder. ¹H NMR(DMSO-d₆, 400 MHz) δ (ppm) 0.91-0.92 (d, J=2.09 Hz, 3H), 1.09 (s, 6H),3.02-3.06 (m, 2H), 3.41 (d, J=6.17 Hz, 2H), 3.53-3.57 (m, 1H), 3.84-3.94(m, 3H), 4.03-4.13 (m, 3H), 4.18-4.23 (m, 1H), 4.91-4.94 (t, J=5.48 Hz,1H), 5.06 (s, 1H), 5.23-5.27 (t, J=6.82 Hz, 1H), 5.65-5.67 (m, 1H),5.79-5.87 (m, 1H), 5.90 (s, 1H), 7.09 and 7.16 (2s, 2H), 7.48-7.55 (m,3H), 7.64-7.67 (m, 2H); ¹⁹F NMR (d₆-DMSO, 235 MHz) δ (ppm) −60.79 (s,3F); ³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm) 9.55 and 9.76 (2s); Scan ES⁺ 655(M+H)⁺, λ_(max)=270 nm; HPLC (0-100% ACN over a period of 10 min)t_(R)=5.03 min λ_(max)=271 nm.

Example 28 Preparation of B204, the Hydroxy-tBuSATEN-(n-methyl-n-octyl-amine)phosphosphoramidate Derivative of2′-C-methylcytidine

Synthetic Scheme:

To a solution of compound 8 (See Example 2, Procedure A, Strategy a)(950 mg, 0.89 mmol) in anhydrous carbon tetrachloride (9 ml) was addeddropwise n-methyl-n-octylamine (10 eq, 1.28 g). The reaction mixture wasstirred at room temperature for 3 h and the solvent removed underreduced pressure. The crude mixture was purified on silica gel columnchromatography (eluant: stepwise gradient [0-3%] of methanol inmethylene chloride) to afford the desired protected nucleoside as a foam(88%). This compound was converted into the phosphoramidate prodrug B204(52 mg, 7%) following experimental conditions described in the Example 2Strategy A and isolated as a white lyophilized powder.

¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 0.83 (m, 3H), 0.93-0.94 (d, J=3.75 Hz,3H), 1.10 (s, 6H), 1.22 (s, 10H), 1.44 (m, 2H), 2.56 (d, J=8.2 Hz, 3H),2.88-2.93 (m, 2H), 3.31 (m, 2H), 3.43 (d, J=5.60 Hz, 2H), 3.50-3.53 (m,1H), 3.91-3.93 (m, 3H), 4.04-4.07 (m, 1H), 4.13-4.16 (m, 1H), 4.91 (t,J=5.59 Hz, 1H), 5.06 (s, 1H), 5.23 (m, 1H), 5.65-5.67 (m, 1H), 5.91 (s,1H), 7.08-7.16 (m, 2H), 7.50-7.57 (m, 1H); ³¹P NMR (DMSO-d₆, 162 MHz) δ(ppm) 10.52 and 10.66 (2s); Scan ES⁺ 623 (M+H)⁺, λ_(max)=280.73 nm; HPLC(0-100% ACN over a period of 8 min) t_(R)=6.07 min λ_(max)=274.9 nm.

Example 29 Preparation of B244, the Hydroxy-tBuSATEN,N-(dibutylamine)phosphoramidate Derivative of 2′-C-methylcytidine

Synthetic Scheme:

To a solution of compound 12 (See Example 2, Procedure A, Strategy b)(1.5 g, 1.46 mmol) in anhydrous carbon tetrachloride (15 ml) was addeddropwise dibutylamine (10 eq, 2.5 ml). The reaction mixture was stirredat room temperature for 3 h and the solvent removed under reducedpressure. The crude mixture was purified on silica gel columnchromatography (eluant: stepwise gradient [0-5%] of methanol inmethylene chloride) to afford the desired protected nucleoside as a foam(61%). This compound was converted into the phosphoramidate prodrug B244(21 mg, 4%) following experimental conditions described in the Example 2Strategy B and isolated as a white lyophilized powder. ¹H NMR (DMSO-d₆,400 MHz) δ (ppm) 0.76-0.81 (td, J=2.40 Hz and J=7.43 Hz, 6H), 0.86-0.87(d, J=5.51 Hz, 3H), 1.05 (s, 6H), 1.11-1.19 (m, 4H), 1.33-1.39 (m, 4H),2.80-2.87 (q, J=9.50 Hz, J=8.67 Hz, 4H), 3.01-3.04 (t, J=6.23 Hz, 2H),3.42-3.43 (m, 2H), 3.50-3.60 (m, 1H), 3.81-3.88 (m, 3H), 3.97-4.01 (m,1H), 4.07-4.10 (m, 1H), 4.84-4.87 (m, 1H), 5.06 (s, 1H), 5.23 and 5.29(2d, J=8.0 Hz, 1H), 5.70 (s, 1H), 5.91 (brs, 1H), 7.10 and 7.17 (2s,2H), 7.49 and 7.55 (2d, J=8.0 Hz, 1H); ³¹P NMR (DMSO-d₆, 162 MHz) δ(ppm) 10.44 and 10.56 (2s); Scan ES⁺ 609 (M+H)⁺, λ_(max)=279.7 nm; HPLC(0-100% ACN over a period of 8 min) t_(R)=5.59 min λ_(max)=274.9 nm.

Example 30 Preparation of B308, the Hydroxy-tBuSATEN-methylbenzylphosphosphoramidate Derivative of 2′-C-methyl-cytidine

Synthetic Scheme:

To a solution of compound 12 (See Example 2, Procedure A, Strategy b)(2.7 g, 2.6 mmol) in anhydrous carbon tetrachloride (26 ml) was addeddropwise N-benzylmethylamine (5 eq, 1.67 ml). The reaction mixture wasstirred at room temperature for 3 h and the solvent removed underreduced pressure. The crude mixture was purified on silica gel columnchromatography (eluant: stepwise gradient [0-5%] of methanol inmethylene chloride) to afford the desired protected nucleoside as a foam(44%). This compound was converted into the phosphoramidate prodrug B308(43 mg, 2%) following experimental conditions described in the Example 2Strategy B and isolated as a white lyophilized powder. ¹H NMR (DMSO-d₆,400 MHz) δ (ppm) 0.93-0.94 (s, 3H), 1.10 (s, 6H), 2.43-2.45 (d, J=4.26Hz, 3H), 3.13 (t, J=6.23 Hz, 2H), 3.36-3.37 (d, J=5.24 Hz, 2H),3.56-3.60 (m, 2H), 3.97-4.01 (m, 3H), 4.07-4.21 (m, 3H), 4.92-4.94 (m,1H), 5.08 (s, 1H), 5.30-5.32 (m, 1H), 5.59-5.67 (2d J=8.0 Hz, 1H), 5.91(s, 1H), 7.13 (m, 2H), 7.42-7.50 (m, 5H), 7.45-7.54 (2d J=8.0 Hz, 1H);³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm) 10.53 and 10.34 (2s); Scan ES⁺ 601(M+H)⁺, λ_(max)=268.7; HPLC (0-100% ACN over a period of 8 min)t_(R)=3.37 min λ_(max)=274.9 nm.

Example 31 Preparation of B353, the Hydroxy-tBuSATEN-piperidinephosphosphoramidate Derivative of 2′-C-methyl-cytidine

Synthetic Scheme:

To a solution of compound 12 (See Example 2, Procedure A, Strategy b)(300 mg, 0.29 mmol) in anhydrous carbon tetrachloride (3 ml) was addeddropwise piperidine (5 eq, 145 μl). The reaction mixture was stirred atroom temperature for 3 h and the solvent removed under reduced pressure.The crude mixture was purified on silica gel column chromatography(eluant: stepwise gradient [0-5%] of methanol in methylene chloride) toafford the desired protected nucleoside as a foam (55%). This compoundwas converted into the phosphoramidate prodrug B353 (19 mg, 22%)following experimental conditions described in the Example 2 Strategy Band isolated as a white lyophilized powder. ¹H NMR (DMSO-d₆, 400 MHz) δ(ppm) 0.92 (d, J=2.56, 3H), 1.10 (s, 6H), 1.44-1.43 (m, 4H), 1.50-1.53(m, 2H), 2.97-3.02 (m, 4H), 3.07-3.10 (t, J=6.66 Hz, 2H), 3.42 (d,J=5.64 Hz, 2H), 3.56-3.60 (m, 1H), 3.89-3.94 (m, 3H), 4.04-4.10 (m, 1H),4.13-4.20 (m, 1H), 4.91-4.93 (t, J=5.64 Hz, 1H), 5.06 (s, 1H), 5.25-5.31(2d, J=9.31 Hz, 1H), 5.68 (m, 1H), 5.90 (s, 1H), 7.17 and 7.10 (2s, 2H),7.50-7.55 (2d, f=9.01 Hz, 1H); ³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm) 8.75and 8.59 (2s); Scan ES⁺ 565 (M+H)⁺, λ_(max)=275.7 nm; HPLC (0-100% ACNover a period of 6 min) t_(R)=3.08 min λ_(max)=273.7 nm.

Example 32 Preparation of B354, the Hydroxy-tBuSATEN-cyclohexylaminephosphosphoramidate Derivative of 2′-C-methyl-cytidine

Synthetic Scheme:

To a solution of compound 12 (See Example 2, Procedure A, Strategy b)(300 mg, 0.29 mmol) in anhydrous carbon tetrachloride (3 ml) was addeddropwise cyclohexylamine (5 eq, 170 μl). The reaction mixture wasstirred at room temperature for 3 h and the solvent removed underreduced pressure. The crude mixture was purified on silica gel columnchromatography (eluant: stepwise gradient [0-5%] of methanol inmethylene chloride) to afford the desired protected nucleoside as a foam(55%). This compound was converted into the phosphoramidate prodrug B354(44 mg, 50%) following experimental conditions described in the Example2 Strategy B and isolated as a white lyophilized powder. ¹H NMR(DMSO-d₆, 400 MHz) δ (ppm) 0.92 (d, J=2.56, 3H), 1.10 (s, 6H), 1.13 (m,5H), 1.46-1.47 (m, 1H), 1.62 (m, 2H), 1.76-1.78 (m, 2H), 2.80 (m, 1H),3.07-3.10 (t, J=6.66 Hz, 2H), 3.42 (d, J=5.64 Hz, 2H), 3.56-3.60 (m,1H), 3.89-3.94 (m, 3H), 4.04-4.10 (m, 1H), 4.13-4.20 (m, 1H), 4.91-4.93(t, J=5.64 Hz, 1H), 5.06 (m, 2H), 5.25 and 5.31 (2d, J=7.2 Hz, 1H),5.68-5.71 (m, 1H), 5.90 (s, 1H), 7.19 and 7.09 (2s, 2H), 7.50 and 7.55(2d, J=7.2 Hz, 1H); ³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm) 9.05 and 8.91(2s) Scan ES⁺ 579 (M+H)⁺, λ_(max)=280.7 nm; HPLC (0-100% ACN over aperiod of 6 min) t_(R)=3.23 min λ_(max)=274.9 nm.

Example 33 Preparation of B391, the Hydroxy-tBuSATEN-morpholinophosphosphoramidate Derivative of 2′-C-methylcytidine

Synthetic Scheme:

To a solution of compound 12 (See Example 2, Procedure A, Strategy b)(350 mg, 0.34 mmol) in anhydrous carbon tetrachloride (3.4 ml) was addeddropwise morpholine (10 eq, 300 μl). The reaction mixture was stirred atroom temperature for 3 h and the solvent removed under reduced pressure.The crude mixture was purified on silica gel column chromatography(eluant: stepwise gradient [0-5%] of methanol in methylene chloride) toafford the desired protected nucleoside as a foam (70%). This compoundwas converted into the phosphoramidate prodrug B391 (53 mg, 49%)following experimental conditions described in the Example 2 Strategy Band isolated as a white lyophilized powder. ¹H NMR (DMSO-d₆, 400 MHz) δ(ppm) 0.92 (d, J=2.56, 3H), 1.10 (s, 6H), 3.0 (m, 4H), 3.07-3.10 (t,J=6.66 Hz, 2H), 3.31 (s, 2H), 3.42 (d, J=5.64 Hz, 2H), 3.56-3.60 (m,3H), 3.89-3.94 (m, 3H), 4.04-4.10 (m, 1H), 4.13-4.20 (m, 1H), 4.91-4.93(t, J=5.64 Hz, 1H), 5.08 (s, 1H), 5.25-5.31 (m, 1H), 5.68-5.71 (d, J=7.2Hz, 1H), 5.90 (s, 1H), 7.18 and 7.12 (2s, 2H), 7.52 and 7.50 (2d, J=7.6Hz, 1H); ³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm) 7.76 and 7.61 (2s); Scan ES⁺567 (M+H)⁺, λ_(max)=279.7 nm; HPLC (0-100% ACN over a period of 10 min)t_(R)=3.42 min λ_(max)=273.7 nm.

Example 34 Preparation of B395, the Hydroxy-tBuSATEN-pyrrolidinephosphosphoramidate Derivative of 2′-C-methylcytidine

Synthetic Scheme:

To a solution of compound 12 (See Example 2, Procedure A, Strategy b)(500 mg, 0.49 mmol) in anhydrous carbon tetrachloride (5 ml) was addeddropwise pyrrolidine (5 eq, 200 μl). The reaction mixture was stirred atroom temperature for 3 h and the solvent removed under reduced pressure.The crude mixture was purified on silica gel column chromatography(eluant: stepwise gradient [0-5%] of methanol in methylene chloride) toafford the desired protected nucleoside as a foam (87%). This compoundwas converted into the phosphoramidate prodrug B395 (48 mg, 21%)following experimental conditions described in the Example 2 Strategy Band isolated as a white lyophilized powder. ¹H NMR (DMSO-d₆, 400 MHz) δ(ppm) 0.93-0.94 (d, J=3.75 Hz, 3H), 1.10 (s, 6H), 1.78-1.79 (q, J=5.80Hz, 4H), 3.09-3.09 (m, 6H), 3.42 (s, 2H), 3.57-3.59 (m, 1H), 3.92-3.93(m, 3H), 4.09-4.11 (m, 1H), 4.16-4.18 (m, 1H), 4.93 (brs, 1H), 5.10 (s,1H), 5.28-5.32 (t, J=8.00 Hz, 1H), 5.70 (d, J=8.0 Hz, 1H), 5.89 (s, 1H),7.27 and 7.40 (2s, 2H), 7.55 and 7.61 (2d, J=8.0 Hz, 1H); ³¹P NMR(DMSO-d₆, 162 MHz) δ (ppm) 7.56 and 7.69 (2s); Scan ES⁺ 551 (M+H)⁺,λ_(max)=275.7 nm; HPLC (0-100% ACN over a period of 10 min) t_(R)=3.88min λ_(max)=273.7 nm.

Example 35 Anti-HBV Activity

The compound of Example 1 (NM 204) (Hydroxy-tBuSATEN-benzylphosphoramidate derivative of L-ddA) (A550) was contacted withHBV-infected HepG2 cells. EC₅₀ values were measured according tostandard techniques. As shown in the table below, the compound ofExample 1 showed significant activity compared to parent molecule LddA.

HBV wt (HepG2) Drug N EC₅₀ (μM) LddA 3 >10 Ex 1 (A550) 3 0.062 ± 0.018LdT 3  0.26 ± 0.048 Lamivudine 3 0.022 ± 0.007

Example 36 Preparation of Calibration Curve for Measurement of ddATP

Measurements of the concentration of2′-3′-dideoxyadenosine-5′-triphosphate (ddATP) (the triphosphatenucleotide of 2′-3′-dideoxyadenosine (ddA) are performed by liquidchromatography tandem mass spectrometry (LC/MS/MS), e.g., of methanolicextracts of hepatocytes.

The concentration of ddATP is measured by comparison to a standardcurve.

Working stock solutions of TP-ddA are prepared from a 100 pmol/μl stocksolution in de-ionized water of ddATP (tetrasodium salt of >91% purity)purchased from Sigma Chemical Co as follows:

ddATP Working Stock Solutions and Preparation of Standard Curve forddATP.

Stock Test conc Vol DlH₂O Total Conc mol com- pmol/ taken vol vol pmol/Per pound μl μL μL μL μl 10 μl 1. Working stock#1 TP-ddA 100 2000 20004000 50.0 500 Stock conc Vol DlH₂O Total Conc Test pmol/ taken vol volpmol/ article μL μL μL μL μL 2. Working stock#2 TP-ddA 100 1000 30004000 25.0 250 3. Working stock #4 (prepared from stock#1) TP-ddA 100 500 3500 4000 12.5 125 4. Working stock #5 (prepared from stock#1)TP-ddA 100  200 3800 4000  5.0  50 5. Working stock #6 (prepared fromstock#1) TP-ddA 100  100 3900 4000  2.5  25 6. Working stock #7(prepared from stock#1) TP-ddA 100  40 3960 4000  1.0  10

Internal standard (ISTD) working stock are prepared from a 0.50 mg/mLstock solution of 2-deoxyadenosine 5-triphosphate purchased from SigmaChemical Co.

Stock conc Vol taken MeOH vol Total vol Conc Conc ISTD μg/mL μL μL μLμg/mL pmol/mL dATP 500 200 9800 10000 10 500

In some embodiments, calibration standards are prepared as follows usingliver

Preparation of cal stds: work- work- std ing ing conc liver work- stockstock ISTD MeOH cal pmol/ wt ing con vol vol vol total std# μl G stock#pmol/μl μL μL μL vol μL Blk   0 0.1  0 50 940  990 #1  50 0.1 #5  5.0 1050 940 1000 #2  125 0.1 #4  12.5 10 50 940 1000 #3  250 0.1 #3  25.0 1050 940 1000 #4  500 0.1 #2  50.0 10 50 940 1000 #5 1000 0.1 #1 100.0 1050 940 1000 samples:

In some embodiments the following HPLC conditions are used for the HPLCMS, e.g. HPLC Tandem MS analysis instrument method:

HPLC is conducted on Phenomenex Luna Amino 3 μm 100A, 30×2 mm column,with a mobile phase: A: 70% 10 mM NH4OAc 30% ACN pH 6.0; and B: 70% 1 mMNH4OAc 30% ACN pH 10.5 as follows:

Gradient elution program: Time Flow Step (min) (μl/min) A (%) B (%) 0 0   400 60 40 1  1.1  400 60 40 2  1.11 400 40 60 3  2.11 400 30 70 4 2.6  400 20 80 5  3.1  400 0 100 6  5.5  400 0 100 7  5.51 400 60 40 810   400 60 40 Injection volume: 50 μl Flow rate to MS: 0.400 mL/min, nosplitting of flow Multiple Reaction Monitoring (MRM) conditions:(API3000) Ionization Mode: Positive Ion Electrospray (ESI+) IonSprayVoltage (IS): 5000 V Temperature (TEM): 550° C. Turbo IS gas 8 L/minNebulizer (NEB): 14 CAD Gas Setting (CAD):  6 Declustering potential  68V (DP) Collision energy (CE) 27 eV Entrance/Exit potentials 10V/11V(EP/CXP) Precursor Product Compound ion => Ion ddA triphosphate 476.2 =>135.9 ddA diphosphate 396.2 => 135.9 dA triphosphate (ISTD) 460.2 =>135.9 *Luna Amino column is directly connected on the inlet end to a“Security Guard” cartridge holder suitable for 2.1 mm Phenomenexcolumns, containing a C18 cartridge.

Example 37 In Vitro Phosphorylation in Hepatocytes

Primary hepatocytes (Rat, Cynomolgus Monkey or human) were seeded at0.8×10⁶ in a collagen-coated 12-well plate and allowed to attach 4-6hours after which time the seeding medium was replaced with serum-freeculture medium and cells allowed to acclimatize to the new mediumovernight. On the next day, cells were exposed for 1, 4, 8 and 24 hoursto test article (NM204) (A550) at 10 and 50 μM prepared in fresh culturemedium from stock solution in DMSO (final DMSO concentration was 0.1%).At each time point, an aliquot (500 μl) was collected and immediatelyadded to 500 μl of acetonitrile and stored at −20° C. until analysis.The remaining exposure medium was removed and the cell monolayer (stuckto dish) washed 2 times with ice-cold PBS. Any remaining PBS wascarefully removed by aspiration and cells were harvested by scraping in1 mL 70% ice-cold methanol. Cell samples were placed overnight at −20°C. and cellular debris removed by centrifugation on the next day. Thesupernatants were removed and filtered prior to analysis by LC/MS. Astandard curve was prepared by using untreated cells processed similarlyexcept that prior to harvesting in 70% methanol, 10 μl of LddATPstandard solutions prepared in methanol were added to the washedmonolayers. These control samples were then processed and analyzed asdescribed for test samples.

The results are shown below:

LddA-TP formation in hepatocytes Ex 1 (A550) 10 μM LddA TP Levels(pmol/million cells) Time (hour) Rat Monkey Human 1 159.5  287.5 161.5 4388.0  978.0 312.5 8 468.5 1230.0 352.5 24 422.0  344.0 366.0 Ex 1(A550) 50 μM LddATP Levels (pmol/million cells) Time (hour) Rat MonkeyHuman 1 393.0 2085.0 682.5 4 1212.0 5690.0 1480.0 8 1590.0 6030.0 1930.024 1505.0 3030.0 2062.5

As indicated from the data, significant levels of L-ddATP were detectedin the hepatocytes. In monkey hepatocytes, the levels appear to reach amaximum level at 8 hours followed by a rapid decline. In contrast,levels in both rat and human hepatocyte appear to level off after 8hours.

Example 38 In Vivo Studies in Rat

Distribution of NM-204 (the compound of Example 1 (Hydroxy-tBuSATEN-benzylphosphoramidate derivative of L-ddA) (A550) in the rat liver wasevaluated following a single intravenous (I.V.) or oral administrationof A550 (NM-204) at a dose of 20 (oral) or 10 (I.V.) mg/Kg body weight.The dose solutions were prepared on the same day prior to doseadministration.

At the specified time point (1 and 3 hours for IV animals or 1, 3 and 8hours for oral animals), each animal was euthanized by CO₂ gas followedby exsanguination via the abdominal vein. Livers were collectedimmediately after sacrifice, flash frozen in liquid nitrogen, placed ondry ice, and later stored at −70° C., before being analyzed.

Preparation of Calibration Standards from Control Liver Extracts:

Control rat liver samples were taken from whole frozen livers(Bioreclamation, Inc. Hicksville, N.Y.) with the aid of a tissue coringutensil (Harris Unicore, 8.0 mm, VWR). Each ˜0.1 g sample was placed inindividual 2 mL poly vials with 0.940 mL of 80% MeOH/20% DIH₂O andhomogenates were prepared using a mechanical tissue disruptor (TissueMaster, Omni-International, Inc, Marietta Ga.). The vials received a 10μl aliquot of a working stock solution and a 50 μl aliquot of the ISTDbefore vortexing for ˜30 sec. The mixtures were stored overnight at −20°C. and the next day were removed for 10 min of centrifugation in abenchtop centrifuge. Each supernatant was transferred to individualcentrifugation filtration units (0.45 μm) and the resulting filtrateswere transferred to HPLC vials for the LC/MS/MS analysis. The finalconcentrations of ddATP in the calibration standards was 1000, 500, 250,125, 50, and 0 pmol/ml. Each calibration standard was directly injectedin a 50 μL volume onto the ion-exchange column for analysis. Standardcurve analysis of calibration standards from control liver extracts wasconducted.

Analysis of ddATP was done by an ion-exchange chromatography method withon-line positive ionization ESI-MS/MS detection in multiple reactionmonitoring (MRM) detection mode. The peak areas obtained for 4 of the 5calibrants allowed for construction of a standard curve thatdemonstrated good linearity (R²=0.9996) over a 50-1000 pmol/mlconcentration range. This is equivalent to a range of 5-100 pmol pergram liver by the sample preparation employed. The HPLC MS MS conditionsdescribed in Example 5 were utilized. The lower limit of quantitationdemonstrated by the LC/MS/MS method is e.g., ˜0.2 pmol/mL for hepatocytecellular extracts which contain much less salt.

The results showing intracellular levels of A550 (NM204) (showing thecompound entered the liver cells) and LddATP (showing cleaving of thephosphoramidate moiety and triphosphorylation of the ddA to the activetriphosphate in the liver) are shown below:

A550 (Ex 1) and LddATP measured in livers of male rats dosed IV or Owith A550 (Ex 1) Concentration Concentration. Compound Time- ddA-TP(A550) (Ex 1) point (pmol/g (pmol/10⁶ Animal Number (pmol/g liver) (hrs)liver) cells)* IV dose (10 mg/kg) 1M1  65.8 1 2025 17.8 1M2  89.1 1 193016.9 1M3  85.1 1 1355 11.9 Mean  80.0 1770 15.5 IV dose (10 mg/kg) 2M1 28.3 3 1345 11.8 2M2  26.0 3 1940 17.0 2M3  29.3 3 2990 26.2 Mean  27.92092 18.3 Oral dose (20 mg/kg) 3M1 411   1  210  1.8 3M2 272   1  575 5.0 3M3  70.2 1  400  3.5 Mean 251    395  3.5 Oral dose (20 mg/kg) 4M1360   3  200  1.8 4M2  92.1 3  330  2.9 4M3 161   3  405  3.6 Mean 204   312  2.7 Oral dose (20 mg/kg) 5M1  16.4 8  280  2.5 5M2  28   8  805 5.2 5M3  16.2 8  275  2.4 Mean  20.1  382  3.3 *Hepatocellularitynumber for rat was 114 × 106 cells per gram liver (Toxicology in Vitro20(2005) 1582-1586.

Thus, these results show that the compound can be used to enhanceconcentration of the drug in the liver. These results also show theenhanced concentration of the active triphosphate which is formed in theliver cells.

Example 39 HCV Replicon Assay

Huh-7 cells containing HCV Con1 subgenomic replicon (GS4.1 cells), (C.Seeger; Fox Chase University, Philadelphia, Pa., USA), are grown inDulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetalbovine serum (FBS), 2 mM L-glutamine, 110 mg/L sodium pyruvate, 1×non-essential amino acids, 100 U/mL penicillin-streptomycin and 0.5mg/mL G418 (Invitrogen). For dose-response testing, the cells are seededin 96-well plates at 7.5×10³ cells/well in a volume of 50 μL andincubated at 37° C./5% CO₂. Three hours after plating, 50 μL of ten2-fold serial dilutions of compounds (highest concentration, 75 μM) areadded and cell cultures were incubated at 37° C./5% CO₂ in the presenceof 0.5% DMSO. Alternatively, compounds are tested at a singleconcentration of 15 μM. In all cases, Huh-7 cells lacking the HCVreplicon served as negative control. The cells are incubated in thepresence of compounds for 72 hours after which they were monitored forexpression of the NS4A protein by enzyme-linked immunosorbent assay(ELISA). For this, the plates were then fixed for 1 min with 1:1acetone:methanol, washed twice with phosphate-buffered saline (PBS),0.1% Tween 20, blocked for 1 hour at room temperature with TNE buffercontaining 10% FBS and then incubated for 2 h at 37° C. with theanti-NS4A mouse monoclonal antibody A-236 (ViroGen) diluted in the samebuffer. After washing three times with PBS, 0.1% Tween 20, the cells areincubated 1 hour at 37° C. with anti-mouse immunoglobulin G-peroxidaseconjugate in TNE, 10% FBS. After washing as described above, thereaction is developed with O-phenylenediamine (Zymed). The reaction isstopped after 30 minutes with 2 NH₂SO₄ and the absorbance is read at 492nm using a Sunrise Tecan spectrophotometer. EC₅₀ values are determinedfrom the % inhibition versus concentration data using a sigmoidalnon-linear regression analysis based on four parameters with TecanMagellan software. When screening at a single concentration, the resultsare expressed as % inhibition at 15 μM. For cytotoxicity evaluation,GS4.1 cells are treated with compounds as described above and cellularviability was monitored using a Cell Titer 96 AQ_(ueous) One SolutionCell Proliferation Assay (Promega). CC₅₀ values are determined from the% cytotoxicity versus concentration data with Tecan Magellan software asdescribed above.

Results

Compounds presented in the table below were assayed according to thereplicon assay described above.

HCV ELISA 2 Compound Reference Structure EC₅₀ (μM) CC₅₀ (μM) NUCLEOSIDEPARENT: A634 (NM107)

++ + EXAMPLE 2: B102

++ + EXAMPLE 26: B187

++ + EXAMPLE 27: B399

++ + EXAMPLE 28: B204

++ + EXAMPLE 29: B244

+ + EXAMPLE 30: B308

++ + EXAMPLE 31: B353

+ + EXAMPLE 32: B354

++ + EXAMPLE 33: B391

+ + EXAMPLE 34: B395

++ + EXAMPLE 24: B234

++ + EXAMPLE 23: B302

++ + EXAMPLE 22: B390

+ + EXAMPLE 25: B183

+ + NUCLEOSIDE PARENT: A844 (NM108)

++ + EXAMPLE 3: B299

+++ + EXAMPLE 11: B242

+++ + EXAMPLE 10: B307

+++ + NUCLEOSIDE PARENT: A374 (NM80)

+++ ++ EXAMPLE 6: B263

++ + NUCLEOSIDE PARENT: C809 (NM106)

+ + EXAMPLE 7: B229

++ + NUCLEOSIDE PARENT: A608

+ + EXAMPLE 8: B186

++ + NUCLEOSIDE PARENT: A849

+++ + EXAMPLE 9: B396

+++ ++ NUCLEOSIDE PARENT: D961

+ + EXAMPLE 12: B503

++ + NUCLEOSIDE PARENT: E810

+++ + EXAMPLE 18: B436

++ + EC₅₀ in ELISA 2 assay is provided as follows: +++ ≦1 μm, ++ >1-10μm and + >10 μm CC₅₀ is provided as follows: ++ ≦75 μm, + >75 μm

Example 40 HBV Drug Susceptibility Assay

-   -   a) Collagen-I coated cell culture plates were seeded with cells        at a density of 0.25-0.5×10⁶ cells per well in 2 ml of        growth/selection media.    -   b) Drug stock solutions were made up freshly in 100% DMSO as        200× stocks. Seven 4-fold dilutions of test compound were        prepared ranging from 2.5 μM to 0.0006 μM (final). Master drug        dilutions were divided into 4 aliquots, and then stored at −20°        C., until used.    -   c) One day after cells were seeded, drug treatments were        initiated by adding 10 μl of drug dilution along with 2 ml of        fresh growth/selection media. Thus, the final DMSO concentration        did not exceed 0.5%. The no-drug control wells received 10 of        DMSO in fresh media.    -   d) Cells were treated every-other-day with 2 ml of fresh drug        combinations/medium for a total of 8 days. Cell lysates were        then collected on day 10 as described below and endogenous        polymerase assay were performed.        Preparation of Nucleocapsid-Containing Lysates for EPA Analysis    -   a) Two days after the final drug treatment, cells were        harvested.    -   b) Media was carefully aspirated and the cell monolayers were        rinsed once with 1 ml of PBS.    -   c) 1 ml of lysis buffer (50 mM Tris-HCl pH 7.5/150 mM NaCl/5 mM        MgCl₂/0.2% NP-40) was added to each well. The detergent is        required to strip the outer envelope from virions and to allow        capture of the inner nucleocapsids. Plates were kept on ice        for >30 min.    -   d) Lysed cells were transferred to 1.5 ml-microfuge tubes.    -   e) Lysates were clarified by spinning at room temperature for 5        min at 14,000 rpm.    -   f) Clarified lysates were transferred to fresh tubes and        immediately frozen on dry-ice, then stored at −80° C. until        endogenous polymerase assays can be performed as described        below.        Endogenous Polymerase Assay (EPA) of Cell Lysates    -   a) EPAs were performed essentially as described in Seifer, et al        (1998). J. Virol. 72: 2765-2776. Clarified lysates were thawed        at room temperature.    -   b) Intracellular HBV nucleocapsids were immunoprecipitated from        the cytoplasmic lysates overnight at 4° C. with a polyclonal        rabbit anti-HBcAg antibody and immobilized on protein A        sepharose CL-4B beads.

c) After 2 washes of the immobilized capsids with 1 ml of EPA washbuffer (75 mM NR₄Cl, 50 mM Tris-HCl pH 7.4, 1 mM EDTA), endogenouspolymerase reactions were initiated by adding 50 μl ofdetergent-containing EPA cocktail (50 mM Tris-HCl pH7.4, 75 mM NH₄Cl, 1mM EDTA, 20 mM MgCl₂, 0.1 mM β-ME, 0.5% NP-40, 100 μM cold dGTP, TTP,dCTP, and 50 nM ³³P-dATP) and incubated overnight at 37° C. Thedetergent is required to enhance permeability of the nucleocapsids.

-   -   d) Following digestion with 1 mg/ml of Proteinase K for 1 hour        at 37° C., endogenously ³³P-labeled HBV DNA was liberated via        phenol/chloroform extraction.    -   e) The nucleic acids were then precipitated with one volume of 5        M NH₄-acetate and 2.5 volumes 100% EtOH, and separated on a 1%        native agarose gel in Tris-borate buffer.    -   f) Gels were blotted onto positively charged nylon membrane        overnight at room temperature via capillary transfer in 0.4 N        NaOH.    -   g) Dried membranes were exposed to a phosphorimager screen (GE        Healthcare) overnight at room temperature, then scanned (Storm        860, GE Healthcare) and quantitated with ImageQuant software (GE        Healthcare).    -   h) Dose-response curves were generated using XLfit 4.1 software.        The mean effective drug concentrations that inhibit endogenous        HBV polymerase activity by 50% were calculated from several        independent experiments.        Cytotoxicity Determination

A standard in vitro cytotoxicity assay was performed in HepG2 cells.Cells were exposed to drug for 9 days. Cell viability was determined viaMTS staining using a CellTiter 96 Aqueous One Solution cellproliferation assay according to the manufacturer's instructions.

-   -   a) HepG2 cells were seeded in 96-well tissue culture plates in        100 μl fresh growth media at 7×10³ cells per well.    -   b) Drug stock solutions were made up in 100% DMSO as 400× stock        solutions and stored at −20° C. until used.

c) Four hours after cells were plated the drug dilutions were preparedand then added to the cells. Cells received up to 100 μM of drug in atotal of 200 μl of fresh growth media containing 0.25% DMSO. Controlwells received growth media with 0.25% DMSO growth media. Plates wereincubated at 37° C., 5% CO₂.

-   -   d) Cells were treated every-other-day with fresh growth media        and fresh drug dilutions for a total of 8 days as described        above.    -   e) On day 9, cell viability of HepG2 cells was determined by        adding 20 μl of MTS CellTiter 96 Aqueous One Solution. Following        4 hours of incubation at 37° C., absorbance was measured at A₄₉₀        nm in a Victor V plate reader (Perkin Elmer).    -   f) The CC₅₀ concentrations were determined using XLfit 4.1        software.

The antiviral in vitro activity of PMEA, B261 (hydroxy-tBuSATEN-benzylphosphoramidate derivative of PMEA as shown in Example 10 Table)along with LdT as control was tested in a total of 4 HBV drugsusceptibility assays. The table below provides the results:

HBV Cell Assay (EPA Read-out) Cytotoxicity Antiviral Activity * Drug(CC₅₀ in μM) (EC₅₀ in μM) SI PMEA >100 0.328 ± 0.082 >310 B261 19.60.016 ± 0.004 1,225 LdT >100 0.366 ± 0.056 >273 Cytoxicity and efficacywas determined on collagen plates.

Example 41 Determination of Total Metabolism in Liver SubcellularFractions Depletion of Parent

NADPH Incubations. Microsomal or S9 incubations were conducted in afinal volume of 0.5 mL. Pooled liver microsomal or S9 protein (1.0mg/mL), suspended in incubation buffer (100 mM potassium phosphate, pH7.4, 5 mM MgCl₂, and 0.1 mM EDTA) was preincubated for 5 min at 37° C.with 10-50 μM OHSATE phosphoramidate compound from a stock solution inDMSO (final DMSO concentration was 0.1%); the reaction was initiated bythe addition of NADPH (3 mM final concentration). Incubations with noNADPH served as controls. At specific times (0-120 min), 0.1 mL sampleswere taken and the reaction terminated by the addition of 1 volume ofstop solution (acetonitrile). The samples were vortex for 30 sec andthen centrifuged at 1500 g for 10 min. The supernatant was transferredto HPLC glass vials and analyzed without further processing by HPLC.FIGS. 1 and 2 depict depletion of NM 108 SATE phosphoroamidate (B299)and NM107 SATE phosphoroamidate (B102), respectively, after incubationwith NADPH in monkey liver S9.

HPLC System for Medium Samples-Unchanged Prodrug

HPLC: Agilent 1100 Column: Phenomenex Luna C18(2), 20 × 2 mm, Mobilephases (MP): MP(A) 10 mM K₂HPO₄ pH5, MP(B) ACN Gradient elution: 20 to63% MP(B) run from 0 to 30 min Runtime: 20 min Flow rate:  1 mL/minInjection volume: 10-20 μL UV: 252 nm-NM108SATE deriv (B299) 272nm-NM107SATE deriv (B102)

Thus, without being limited to any theory, since the metabolism is NADPHdependent, it is possible that the phosphoroamidate compound ispreferentially activated by Cytochrome P450 in the liver.

Example 42 Determination of Triphosphate Levels in Cells

Preparation of Primary Hepatocyte Cultures

Freshly isolated cells from animal and human liver were obtained insuspension on ice. Following receipt, cells were pelleted bycentrifugation at 500 rpm (rat) or 700 rpm (monkey and human) andresuspended at 0.8 million cells per mL of platting medium (HPM).Multi-well collagen-coated plates (12-well) were then seeded by additionof 1 mL of cell suspension (0.8 million cells/mL). The plates weregently shaken to evenly distribute the cells and placed in an incubatorat 37° C. for approximately 4 to 6 hours to allow cells to attach. Oncecells have attached, the platting medium was removed and replaced withhepatocyte culture medium (HCM). Cells were left overnight in anincubator at 37° C. to acclimatize to culture and the medium.

Incubations with Test Article

Hepatocyte incubations were conducted in a final volume of 1.0 mLHCM/well (0.8 million cells/mL). HCM from overnight incubation of cellswas removed and replaced with fresh HCM, pre-warmed to 37° C.,containing 10 μl M test article from a stock solution in DMSO (finalDMSO concentration was 0.1%). At specific times (up to 24 hrs),incubation medium was discarded and the cell monolayers were carefullywashed two times with ice-cold PBS. Following the last wash, all PBS wascarefully removed and 1 mL of extraction buffer (ice-cold 70% methanol)was added. Each well was sealed with parafilm immediately followingaddition of methanol. Once the entire plate was processed, additionalparafilm was placed on entire plate forming a double seal to preventevaporation during the extraction process. The cover lid was then placedon the plate and sealed with tape. The plates were then stored at −20°C. for a minimum of 24 hrs to allow for extraction of intracellularcontents.

Preparation of Huh7 or HepG2 Cultures

HepG2s or Huh7 cells were plated at 0.4×10⁶ cells/well incollagen-coated 12-well plates. Cells were allowed to attach overnight.Culture medium from overnight incubation of cells was removed andreplaced with fresh culture medium, pre-warmed to 37° C., containing 10μM test article from a stock solution in DMSO (final DMSO concentrationwas 0.1%). After 24-72 hours, incubation medium was discarded and thecell monolayers were carefully washed two times with ice-cold PBS.Following the last wash, all PBS was carefully removed and 1 mL ofextraction buffer (ice-cold 70% methanol) was added. Each well wassealed with parafilm immediately following addition of methanol. Oncethe entire plate was processed, additional parafilm was placed on entireplate forming a double seal to prevent evaporation during the extractionprocess. The cover lid was then placed on the plate and sealed withtape. The plates were then stored at −20° C. for a minimum of 24 hrs toallow for extraction of intracellular contents.

Sample Preparation for Analysis

Cellular extracts were prepared by transferring 0.9 mL of extract into 2mL microfuge tubes followed by centrifugation for 5 min at 14,000 rpm.Approximately 100 μL of the supernatant was transferred to HPLC vialsand triphosphate levels determined by LCMS/MS as described below.

HPLC Conditions: NM107-Triphosphate

HPLC: Column: Phenomenex Luna Amino 3 μM 100A, 30 × 2 mm, (A) 70% 10 mMNH₄OAc 30% ACN pH 6.0 Mobile phases (MP): (B) 70% 1 mM NH₄OAc 30% ACN pH10.5 Gradient elution: Step Time Flow A B 0 0.00 400 80 20 1 0.10 400 8020 2 0.11 400 40 60 3 0.21 400 40 60 4 2.60 400 10 90 5 2.61 400 0 100 65.60 400 0 100 7 5.61 400 80 20 8 9.00 400 80 20 Flow rate to MS: 0.400mL/min, no split Injection volume: 10 μL Compound Precursor ion Production NM107 triphosphate 498.0 112.0

Exemplary HPLC Conditions: NM108-Triphosphate

HPLC: Column: Phenomenex Luna Amino 3 μm 100A, 30 × 2 mm, Mobile phases(MP): (A) 70% 10 mM NH₄OAc 30% ACN pH 6.0 (B) 70% 1 mM NH₄OAc 30% ACN pH10.5 Gradient elution: Step Time Flow A B 0 0.00 400 60 40 1 0.10 400 6040 2 0.11 400 40 60 3 0.21 400 40 60 4 2.60 400 10 90 5 2.61 400 0 100 65.61 400 0 100 7 5.61 400 60 40 8 9.00 400 60 40 Flow rate to MS: 0.400mL/min, no split Injection volume: 10 μL Compound Precursor ion Production NM108 triphosphate 538.0 152.0

NM 107 triphosphate and B102 triphosphate levels in cell extracts wereobserved as follows:

Intracellular Triphosphate (pmol/million cells) drug in culture HumanMonkey HepG2* Huh7* B102 991 1838 1.5 9.2 NM107 19 10 17 37 24 hrincubation in 10 μM drug *72 hr incubation in 10 μM drug

As seen from the data levels of intracellular triphosphate for B102 werehigher compared to those for NM107.

Example 43 Demonstration of Potent Antiviral Activity of SecondGeneration Nucleoside Inhibitors, B102, in HCV-Infected Chimpanzees

Nucleoside analogs such as NM107 (2′ methyl cytidine, valopicitabinenucleoside component) have shown efficacy against HCV in the clinicalsetting and their 5′-triphosphates (TP) can be potent inhibitors of HCVNS5B polymerase. However, their wide systemic distribution andinefficient hepatic conversion to TP may lead to reduced safety andantiviral activity. The in vivo preclinical safety and antiviralactivity of the nucleotide prodrugs, B102 were assessed.

Methods: For pharmacokinetic (PK) and toxicology studies, B102 wereorally administered to rats or monkeys at doses from 20 to 300 mg/kg/dayup to 14 days. Hepatic nucleoside TP levels were determined by LC-MS/MS.Compounds (10 mg/kg/day) were administered once daily by oral gavage for4 days in chimpanzees chronically infected with HCV genotype 1. HCVviral loads were monitored before, during and after treatment byquantitative RT-PCR.

Results: PK studies in rat and monkey revealed that B102 and has afirst-pass hepatic extraction of >95% with low systemic exposure (<1%).Hepatic TP levels were 10-50-fold higher with nucleotide prodrugs versusa nucleoside counterpart. No toxicity was observed after administrationof 50 mg/kg/day of A2 to monkeys for 14 days. No initial emesis or G1toxicity was observed. In HCV-infected chimpanzees, B102 produced arapid and potent antiviral effect followed by a rebound to baselineafter drug discontinuation. Mean viral load reductions ranged from 1.5log 10 with B102 to over 4 days of drug exposure. An equivalent dose ofvalopicitabine led to a 0.7 log 10 viral reduction. No lab abnormalitiesor evidence of toxicities were observed in chimpanzees.

Thus, when orally administered, B102 generates high hepatic levels oftriphosphates coupled with low systemic exposure, leading to rapid andpotent inhibition of HCV replication in chimpanzees, thus demonstratinga promising in vivo preclinical safety profile and antiviral activity.

Example 44

Compounds were tested in an anti-HBV assay. Determination of anti-HBVactivity in HBV virions and nucleocapsids via endoenous polymeraseassays (EPA)

Drug susceptibility assay using a wild-type HBV producer cell line

1. 12-well collagen-I coated plates were seeded with producer cellsexpressing wild-type HBV at a density of 0.5−1×10⁶ cells per well in 2ml growth/selection media.

2. Drug stock solutions were made up freshly in 100% DMSO as 200×stocks. Five additional 4-fold dilutions were prepared from these 200×stock in 100% DMSO. For each experiment, 4 aliquots of each drugdilution series were stored at −20° C. until used.

3. Once cells reached confluency (1 day after cells were seeded), drugtreatment was initiated by adding 10 μl of drug dilution into 2 ml offresh growth/selection media. Thus, the final DMSO concentration did notexceed 0.5%. The no-drug control wells received only 10 μl of DMSO infresh media.4. Cells were treated every-other-day with 2 ml of fresh drug/medium fora total of 8 days. Cell lysates were then collected on day 10 andsubjected to EPA analysis as described below.Preparation of Nucleocapsid-Containing Lysates for EPA Analysis1. Cells were grown for 3 to 4 days in 12-well collagen-I coated platesuntil confluent.2. Media was carefully aspirated and the cell monolayers were rinsedonce with 1 ml of PBS.3. One ml of lysis buffer (50 mM Tris-HCl pH 7.5/150 mM NaCl/5 mMMgCl₂/0.2% NP-40) was added to each well. Plates were stored on ice for30 min to 4 h.4. Lysed cells were transferred to 1.5 ml-microfuge tubes.5. Lysates were clarified by spinning at 14,000 rpm for 5 mM at roomtemperature.6. Clarified lysates were transferred to fresh tubes and immediatelyfrozen on dry-ice, then stored at −80° C. until endogenous polymeraseassays were performed as described below.Preparation of Secreted Virions from Supernatant for EPA Analysis1. Cells were grown for 3 to 4 days in 12-well collagen-I coated platesuntil confluent.2. Media was carefully aspirated and transferred to 1.5 ml-microfugetubes.3. Supernatants were clarified by spinning at 14,000 rpm for 5 min atroom temperature.4. Clarified supernatants were transferred to fresh tubes andimmediately frozen on dry-ice, then stored at −80° C. until endogenouspolymerase assays were performed essentially as described below.Endogenous Polymerase Assay (EPA) of Cell Lysates and Supernatants1. EPAs were performed essentially as described by Seifer et al (1998).Intracellular HBV nucleocapsids were immunoprecipitated from thecytoplasmic lysates overnight at 4° C. with a polyclonal rabbitanti-HBcAg antibody and immobilized on protein A sepharose CL-4B beads.Secreted virions were immunoprecipitated from clarified cellsupernatants overnight at 4° C. with a monoclonal mouse anti-LS antibody(MA 18/7) in the absence of detergent.2. Following 3 washes of the immobilized capsids or virions with 1 ml ofEPA wash buffer (75 mM NH₄Cl, 50 mM Tris-HCl pH 7.4, 1 mM EDTA),endogenous polymerase reactions were initiated by adding 50 μl ofdetergent-containing EPA cocktail (50 mM Tris-HCl pH7.4, 75 mM NH₄Cl, 1mM EDTA, 20 mM MgCl₂, 0.1 mM β-ME, 0.5% NP-40, 100 μM cold dGTP, TTP,dCTP, and 50 nM ³³P-dATP) and incubated overnight at 37° C. Thedetergent is required to strip the outer envelope fromimmunoprecipitated virions as well as to enhance permeability of thenucleocapsids.3. Following digestion with 1 mg/ml of proteinase K for 1 h at 37° C.,endogenously ³³P-labeled HBV DNA was liberated via phenol/chloroformextraction.4. The nucleic acids were precipitated with 1 volume of 5M NIL-acetateand 2.5 volumes of 100% EtOH, and then separated on a 1% native agarosegel in Tris-borate-EDTA buffer.5. Gels were blotted onto positively charged nylon membrane overnight atroom temperature via capillary transfer in 0.4 N NaOH.6. Dried membranes were exposed to a PhosphorImager screen (GEHealthcare) overnight at room temperature, then scanned (Storm 860, GEHealthcare) and quantitated with ImageQuant software (GE Healthcare).7. The 50% effective concentration (EC₅₀) values were calculated fromthe resulting best-fit equations determined by Xlfit, version 4.1(IDBS).

The following results were obtained.

Compound Virion RI Ref. No. Structure EC₅₀ (μM) EC₅₀ (μM) A348 (NM 48)

+++ A362 (NM 77)

++ ++ A616 (NM 128)

++ C819 (NM 177)

++ ++ A361 (NM 55)

+ ++ A550 (NM 204)

+++ +++ C791

++ B261

+++ PMEA

+++ L-dT +++ EC₅₀ in HBV virion and RI is provided as follows: +++ ≦1μm, ++ >1-10 μm and + >10 μm

Example 45 Ethynyl Nucleosides for the Treatment of HCV

Exemplary compound syntheses are described below:

29:{9-[(2R)-2-Deoxy-2-fluoro-2-C-ethynyl-β-D-erythro-furanosyl]-guanin}-5′-yl-O-(triphenylmethyloxy-tert-butyl-S-acyl-2-thioethyl)H-phosphonate

To a stirred solution of 7j (0.32 mmol) and S-(2-Phosphite-ethyl)2,2-dimethyl-3-triphenylmethyloxy-thiopropionate (0.42 mmol) in pyridine(5 ml) at −15° C. was added dropwise pivaloyl chloride (0.64 mmol) undernitrogen. The reaction mixture was stirred at −15° C. for 2 hours.Dichloromethane and NH₄Cl solution were added. Organic phase wasseparated washed with NH₄Cl solution, dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The crude material was purified byflash column chromatography (DCM/MeOH) to yield the title compound Brownpowder. Molecular Formula C₃₈H₃₉FN₅O₈PS NMR (DMSO-d₆, 400 MHz) δ (ppm)1.12 (s, 6H), 1.84 (m, 4H) 3.04 (s, 2H), 3.92 (d, J=5.60 Hz, 1H),4.01-4.10 (m, 3H), 4.33-4.39 (m, 2H), 4.60-4.66 (m, 1H), 6.13 (d,J=18.00 Hz, 1H), 6.67 (s, 2H), 7.21-7.35 (m, 15H), 7.81 (s, 1H), 10.86(brs, 1H)

30a:N-(4-fluoro-benzylaminyl)-{9-[(2R)-2-deoxy-2-fluoro-2-C-ethynyl-β-D-erythro-furanosyl]-guanin}-5′-yl-O-(triphenylmethyloxy-tert-butyl-S-acyl-2-thioethyl)phosphate

To a stirred solution of 29 (0.088 mmol) in anhydrous carbontetrachloride (880 μL), 4-fluoro-benzylamine (0.44 mmol) was addeddropwise. The reaction mixture was stirred at room temperature for 2 hand evaporated to dryness (bath temperature not exceeding 30° C.). Thecrude mixture was filtered on a silica gel plug, eluting with a gradient0-10% methanol in dichloromethane to yield the title compound. Whitesolid. Molecular Formula C₄₅H₄₅F₂N₆O₈PS ¹H NMR (DMSO-d₆, 400 MHz) δ(ppm) 1.09 (s, 6H), 3.03 (s, 2H), 3.39-3.41 (m, 2H), 3.90-3.93 (m, 5H),4.05-4.08 (m, 1H), 4.20-4.23 (m, 2H), 4.62-4.65 (m, 1H), 5.74 (m, 1H),6.08-6.14 (dd, J=17.94 Hz and J=4.22 Hz, 1H), 6.32 (m, 1H), 6.67 (s,2H), 7.21-7.35 (m, 19H), 7.81 (s, 1H), 10.86 (brs, 1H) ³¹P NMR (DMSO-d₆,162 MHz) δ (ppm) 9.83 (s, 1P) ¹⁹F NMR (DMSO-d₆, 235 MHz) δ (ppm) −116.24(s, 1F), −158-0.2 (s, 1F) Scan ES⁺ 899 (M−H)⁺, UV λ_(max) 255 nm

31a:N-(4-fluoro-benzylaminyl)-{9-[(2R)-2-deoxy-2-fluoro-2-C-ethynyl-β-D-erythro-furanosyl]-guanin}-5′-yl-O-(hydroxy-tert-butyl-S-acyl-2-thioethyl)phosphate

30a (0.09 mmol) was dissolved in dichloromethane (320 μL) and treatedwith formic acid (32 μL). The mixture was stirred at room temperaturefor 10 min, filtered through a solid phase extraction column elutingwith a gradient 0-30% methanol in dichloromethane, then purified byreverse phase (C 18) silica gel column chromatography eluting with agradient 0-100% acetonitrile in water and lyophilised from a mixture ofwater/dioxan to yield the title compound. White solid. Molecular FormulaC₂₆H₃₁F₂N₆O₈PS NMR (d₆-DMSO, 400 MHz) δ (ppm) 1.09 (s, 6H), 3.03 (s,2H), 3.39-3.41 (m, 2H), 3.90-3.93 (m, 5H), 4.05-4.08 (m, 1H), 4.20-4.23(m, 2H), 4.62-4.65 (m, 1H), 4.92 (m, 1H), 5.74 (m, 1H), 6.08-6.14 (dd,J=17.94 Hz and J=4.22 Hz, 1H), 6.32 (m, 1H), 6.67 (s, 2H), 7.21-7.35 (m,4H), 7.81 (s, 1H), 10.86 (brs, 1H) ³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm)9.66 (s, 1P) ¹⁹F NMR (DMSO-d₆, 235 MHz) δ (ppm) −116.24 (s, 1F), −158.44(s, 1F) Scan ES⁺ 657 (M−H)⁺, UV λ_(max) 254 nm

30b:N-(4-methoxy-benzylaminyl)-{9-[(2R)-2-deoxy-2-fluoro-2-C-ethynyl-β-D-erythro-furanosyl]-guanin}-5′-yl-O-(triphenylmethyloxy-tert-butyl-S-acyl-2-thioethyl)phosphate

30b was synthesized from 29 and 4-methoxy-benzylamine as described for30a. White solid. Molecular Formula C₄₆H₄₈FN₆O₉PS ¹H NMR (DMSO-d₆, 400MHz) (ppm) 1.09 (s, 6H), 3.03 (m, 2H), 3.42 (d, J=5.02 Hz, 2H), 3.71 (d,J=3.60 Hz, 3!:H), 3.85-3.90 (m, 5H), 4.06-4.10 (m, 1H), 4.23-4.29 (m,2H), 4.60-4.66 (m, 1H), 5.54-5.57 (m, 1H), 6.08-6.14 (dd, J=17.94 Hz andJ=4.22 Hz, 1H), 6.28-6.33 (m, 1H), 6.60 (s, 2H), 6.80-6.85 (m, 2H),7.18-7.20 (m, 2H), 7.23-7.25 (m, 15H), 7.82 (s, 1H), 10.56 (brs, 1H))³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm) 9.83 (s, 1P) ¹⁹F NMR (DMSO-d₆, 235MHz) δ (ppm) −116.24 (s, 1F), −158.2 (s, 1F) Scan ES⁺ 911 (M−H)⁺, UVλ_(max) 255 nm

31b:N-(4-methoxy-benzylaminyl)-{9-[(2R)-2-deoxy-2-fluoro-2-C-ethynyl-β-D-erythro-furanosyl]-guanin}-5′-yl-O-(hydroxy-tert-butyl-S-acyl-2-thioethyl)phosphate

31b was synthesized from 30b as described for 31a. White solid.Molecular Formula C₂₇H₃₄FN₆O₉PS ¹H NMR (d₆-DMSO, 400 MHz) δ (ppm) 1.09(s, 6H), 3.03 (m, 2H), 3.42 (d, J=5.02 Hz, 2H), 3.71 (d, J=3.60 Hz, 3H),3.85-3.90 (m, 5H), 4.06-4.10 (m, 1H), 4.23-4.29 (m, 2H), 4.60-4.66 (m,1H), 4.92 (t, J=5.50 Hz, 1H), 5.54-5.57 (m, 1H), 6.08-6.14 (dd, J=17.94Hz and J=4.22 Hz, 1H), 6.28-6.33 (m, 1H), 6.60 (s, 2H), 6.80-6.85 (m,2H), 7.18-7.20 (m, 2H), 7.82 (s, 1H), 10.56 (brs, 1H) ³¹P NMR (DMSO-d₆,162 MHz) δ (ppm) 9.86 (s, 1P) ¹⁹F NMR (DMSO-d₆, 235 MHz) δ (ppm) −158.24(s, 1F) Scan ES⁺ 669 (M−H)⁺, UV λ_(max) 254 nm

30c:N-(4-trifluoro-benzylaminyl)-{9-[(2R)-2-deoxy-2-fluoro-2-C-ethynyl-β-D-erythro-furanosyl]-guanin}-5′-yl-O-(triphenylmethyloxy-tert-butyl-S-acyl-2-thioethyl)phosphate

30c was synthesized from 29 and 4-trifluoromethyl-benzylamine asdescribed for 30a. White solid. Molecular Formula C₄₆H₄₅F₄N₆O₈PS ¹H NMR(d₆-DMSO, 400 MHz) (ppm) 1.09 (s, 6H), 3.03 (t, J=6.44 Hz, 2H), 3.42 (s,2H), 3.87-3.92 (m, 5H), 4.03-4.08 (m, 1H), 4.24-4.29 (m, 2H), 4.60-4.64(m, 1H), 5.79-5.82 (m, 1H), 6.08-6.14 (dd, J=17.94 Hz and J=4.22 Hz,1H), 6.28-6.33 (m, 1H), 6.60 (s, 2H), 7.23-7.25 (m, 15H), 7.50-7.70 (m,4H), 8.25 (brs, 1H), 10.76 (brs, 1H), ³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm)9.86 (s, 1P) ¹⁹F NMR (DMSO-d₆, 235 MHz) δ (ppm) −158.20 (s, 1F) Scan ES⁺949 (M−H)⁺, UV λ_(max) 254 nm

31c:N-(4-trifluoromethyl-benzylaminyl)-{9-[(2R)-2-deoxy-2-fluoro-2-C-ethynyl-β-D-erythro-furanosyl]-guanin}-5′-yl-O-(hydroxy-tert-butyl-S-acyl-2-thioethyl)phosphate

31c was synthesized from 30c as described for 31a. White solid.Molecular Formula C₂₇H₃₁F₄N₆O₈PS ¹NMR (d₆-DMSO, 400 MHz) δ (ppm) 1.09(s, 6H), 3.03 (t, J=6.44 Hz, 2H), 3.42 (s, 2H), 3.87-3.92 (m, 5H),4.03-4.08 (m, 1H), 4.24-4.29 (m, 2H), 4.60-4.64 (m, 1H), 4.91 (brs, 1H),5.79-5.82 (m, 1H), 6.08-6.14 (dd, J=17.94 Hz and J=4.22 Hz, 1H),6.28-6.33 (m, 1H), 6.60 (s, 2H), 7.50-7.70 (m, 4H), 8.25 (brs, 1H),10.64 (brs, 1H), ³¹P NMR (DMSO-d₆, 162 MHz) δ (ppm) 9.86 (s, 1P) ¹⁹F NMR(DMSO-d₆, 235 MHz) δ (ppm) −158.24 (s, 1F) Scan ES⁺ 669 (M−H)⁺, UVλ_(max) 254 nm

Further exemplary compounds synthesized using procedures similar tothose described herein are listed below. Note the following names forthe compounds synthesized in the examples.

-   6a:    6-Chloro-9-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]purine-   6b:    N2-Isobutyryl-9-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]guanine-   6c:    1-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]uracile-   6d:    1-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]thymine-   6e:    N4-Benzoyl-1-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]cytosine-   6f:    5-Fluoro-1-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]uracile-   6g:    4-Chloro-7-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]pyrrolo[2,3-d]pyrimidine-   7i:    9-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]adenine-   7j:    9-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]guanine-   7k:    1-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]cytosine-   7l:    4-Amino-7-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]pyrrolo[2,3-d]pyrimidine-   11c:    1-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]-uracil-5′-yl-bis(S-pivaloyl-2-thioethylphosphate-   11f:    1-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]-5-fluorouracil-5′-yl-bis(S-pivaloyl-2-thioethylphosphate)-   11k:    1-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]-cytosin-5′-yl-bis(S-pivaloyl-2-thioethylphosphate)-   11l:    1-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]-4-aminopyrrolo[2,3-d]pyrimidin-5′-yl-bis(S-pivaloyl-2-thioethylphosphate)-   16:    9-[(2R)-2,3-Dideoxy-2-C-ethynyl-2-fluoro-β-D-glycero-pentofuranosyl]guanine-   17:    9-[(2R)-2,3-Dideoxy-2-C-ethynyl-2-fluoro-β-D-glycero-pentofuranosyl]guanin-5′-yl-bis(S-pivaloyl-2-thioethylphosphate)-   20:    9-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]guanin-5′-yl-bis(S-pivaloyl-2-thioethylphosphate-   23:    9-[(2R)-2-Deoxy-3,5-di-O-isobutyryl-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]guanine-   24:    N2-Isobutyryl-9-[(2R)-2-Deoxy-3,5-di-O-isobutyryl-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]guanine-   27i:    9-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]adenine    5′-triphosphate sodium salt-   27j:    9-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]guanine    5′-triphosphate sodium salt-   28:    9-[(2R)-2,3-dideoxy-2-C-ethynyl-2-fluoro-β-D-glycero-pentofuranosyl]guanine    5′-triphosphate sodium salt

3a:6-Chloro-9-[2-oxo-3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-β-D-ribo-furanosyl]purine

6-Chloro-[3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-β-D-ribo-furanosyl]purine(18.84 mmol) was coevaporated twice with THF then dissolved in anhydrousTHF (50 mL). Anhydrous DMSO (119.82 mmol) was added and the resultingsolution was cooled down to between −40° C. and −30° C. Trifluoroaceticanhydride (36.17 mmol) was added dropwise and the solution was stirredbetween −40° C. and −30° C. for 2 h after which EtN₃ (97.52 mmol) wasadded. The resulting solution was allowed to warm up to room temperatureover 30 min while stirring, then diluted with diethyl ether and washedwith H₂O, dried (Na₂SO₄) and evaporated to dryness. The crude materialwas purified by column chromatography eluting with 1% ethyl acetate indichloromethane. The yellow oil obtained was dissolved in DCM andstirred with an excess of MgSO₄ at room temperature for 36 h, filteredand concentrated under reduced pressure to give the title compound. Paleyellow foam. Molecular Formula C₂₂H₃₅ClN₄O₅Si₂. ¹H NMR (DMSO-d₆, 250MHz) δ (ppm) 9.01 (s, 1H, H-8), 8.61 (s, 1H, H-2), 6.35 (s, 1H, H-1′),5.35 (d, 1H, H-3′, J_(3′-4′)=9.7 Hz), 4.31 (m, 1H, H-4′), 4.12-4.09 (m,2H, H-5′, H-5″), 1.22-0.94 (m, 28H, iPr). LRFAB-MS (GT): 527 (M+H)⁺, 525(M−H)⁻. UV λ_(max) 263 nm. R_(f) 0.17 (ethyl acetate/CH₂Cl, 10/90, v/v).

4a and 4′ a:6-Chloro-9-[3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisdoxane)-2-C-trimethylsilylethynyl-β-D-arabino-furanosyl]purine(4a) and6-chloro-9-[3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyl-disiloxane)-2-C-trimethylsilylethynyl-β-D-ribo-furanosyl]purine(4′a)

Trimethylsilylacetylene (59.20 mmol) was dissolved in anhydrous THF (70mL). n-Butyllithium (37 mL, 1.6 M in hexanes) was added dropwise at −78°C. The reaction mixture was stirred for 30 min at −78° C. and thenallowed to warm up to −55° C. 3a (11.84 mmol) in solution in THF (34 mL)was added dropwise at −78° C. The reaction mixture was stirred for 1 hat −78° C. and then allowed to warm up to −30° C. The reaction wasquenched by careful addition of aqueous saturated NH₄Cl (45 mL) at −78°C. After warming to room temperature, the mixture was diluted withdiethyl ether, washed with saturated brine, dried (Na₂SO₄) andconcentrated to dryness. The crude material was purified by silica gelchromatography eluting with 20% Et₂O in petroleum ether to yield twocompounds: 4a (4.62 g, 62%). Pale yellow foam. Molecular FormulaC₂₇H₄₅ClN₄O₅Si₃ ¹H NMR (DMSO-d₆, 200 MHz) δ (ppm) 8.81 (s, 1H, 1H), 8.64(s, 1H, H-2), 6.64 (s, 1H, OH-2′), 6.33 (s, 1H, H-1′), 4.57 (d, 1H,H-3′, J_(3′4′)=6.6 Hz), 4.20-3.97 (m, 3H, H-4′, H-5′ and H-5″),1.20-1.00 (m, 28H, iPr), 0.14 (s, 9H, Si(CH₃)₃). LRFAB-MS (GT): 625(M+H)⁺. R_(f) 0.72 (ethyl acetate/CH₂Cl, 10/90, v/v);

and 4′ a (0.75 g, 10%). Yellow oil. Molecular Formula C₂₇H₄₅ClN₄O₅Si₃ ¹HNMR (DMSO-d₆, 200 MHz) δ (ppm) 8.80 (s, 1H, H-8), 8.73 (s, 1H, H-2),6.64 (s, 1H, OH-2′), 6.55 (s, 1H, H-1′), 4.62 (d, 1H, H-3′,³J_(3′-4′)=9.1 Hz), 4.39 (m, 1H, H-4′), 4.13 (dd, 1H, H-5′,J_(5′-4′)=3.4 Hz, ²J_(5′-5″)=13.2 Hz), 3.90 (dd, 1H, H-5″, J_(5″-4′)=2.6Hz, ²J_(5″-5″)=13.2 Hz), 1.15-1.00 (m, 28H, iPr), 0.10 (s, 9H,Si(CH₃)₃). LRFAB-MS (GT): 625 (M+H)⁺. R_(f) 0.64 (ethyl acetate/CH₂Cl,10/90, v/v).

5a:6-Chloro-9-[(2R)-2-deoxy-2-fluoro-3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethylsilylethynyl-β-D-erythro-pentofuranosyl]purine

4a (6.78 mmol) was dissolved in dried toluene (31.8 mL) under argon andcooled to −20° C. DAST (40.68 mmol) was added dropwise and the coolingbath was removed after the addition was complete. Stirring was continuedfor 1.5 hour and the mixture was dissolved with ethyl acetate and pouredinto saturated NaHCO₃ and stirred for 5 min. The organic layer waswashed with saturated brine, dried (Na₂SO₄), concentrated, and purifiedby silica gel chromatography eluting with 20% Et₂O in petroleum ether togive the title compound (1.11 g, 26%). Yellow oil. Molecular FormulaC₂₇H₄₄ClN₄O₄Si₃. ¹H NMR (CDCl₃-d₆, 200 MHz) δ (ppm) 8.79 (s, 1H, H-8),8.48 (s, 1H, H-2), 6.48 (d, 1H, H-1′, J_(1′-F)=16.0 Hz), 4.74 (dd, 1H,H-3′, J_(3′-4′)=9.4 Hz, J_(3′-F)=22.4 Hz), 4.36 (d, 1H, H-5′,²J_(5′-5″)32 13.4 Hz), 4.20 (m, 1H, H-4′), 4.10 (dd, 1H, H-5″,²J_(5″-5′)=13.4 Hz, J_(5″-4′)=2.6 Hz), 1.30-1.10 (m, 28H, iPr), 0.00 (s,9H, Si(CH₃)₃). LRFAB-MS (GT): 627 (M+H)⁺. UV λ_(max) 263 nm. R_(f) 0.24(diethyl ether/petroleum ether, 30/70, v/v).

6a:6-Chloro-9-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]purine

A mixture of 5a (3.65 mmol) and ammonium fluoride (47.45 mmol) inmethanol (12.5 mL) was heated at reflux for 2 h. After cooling down toroom temperature, the mixture was concentrated to dryness and purifiedon silica gel chromatography eluting with a stepwise gradient 2-4% ofmethanol in DCM to provide the title compound (0.89 g, 78%). Yellowsolid. Molecular Formula C₁₂H₁₀ClFN₄O₃. ¹H NMR (DMSO-d₆, 200 MHz) δ(ppm)9.02 (s, 1H, H-8), 8.89 (s, 1H, H-2), 6.55 (d, 1H, H-1′, J_(1′-F)=16.1Hz), 6.34 (ld, 1H, OH-3′), 5.38 (lt, 1H, OH-5′), 4.64 (dt, 1H, H-3′,J_(3′-4′)=9.3 Hz, J_(3′-F)=22.5 Hz), 4.07 (m, 1H, H-4′), 3.83 (m, 2H,H-5′, H-5″), 3.76 (d, 1H, ethynyl, ⁴J_(H-F)=5.3 Hz). ¹³C NMR (DMSO-d₆,75 MHz) δ(ppm) 152.0 (C-2), 151.2 (C-4), 149.5 (C-6), 144.7 (C-8), 130.9(C-5), 95.1 (d, C-2′, ¹J_(2′-F)=182.3 Hz), 88.0 (d, C-1′, ²J_(1′-F)=39.8Hz), 82.9 (d, CCH, J_(C-F)=8.2 Hz), 82.5 (C-4′), 75.3 (d, CCH,J_(C-F)=31.5 Hz), 72.7 (d, C-3′, ²J_(3′-F)=19.5 Hz), 59.0 (C-5′). ¹⁹FNMR (DMSO-d₆, 235 MHz) δ(ppm) −159.0 (m). LC/MS: (M+H⁺) 313.1 (8.29min). UV λ_(max) 262 nm. R_(f) 0.21 (MeOH/CH₂Cl, 7/93, v/v).

7i:9-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]adenine

6a (2.24 mmol) was dissolved in saturated ammoniacal methanol (80 mL)and heated for 4 h in a steel bomb at 90° C. After cooling down to roomtemperature the mixture was coevaporated to dryness and purified bysilica gel chromatography eluting with a gradient 5-8% of methanol inDCM to yield the title compound (305 mg, 46%). Yellow solid. MolecularFormula C₁₂H₁₂FN₅O₃. ¹H NMR (DMSO-d₆, 200 MHz) δ(ppm) 8.40 (s, 1H, H-8),8.17 (s, 1H, H-2), 7.38 (ls, 2H, NH₂) 6.35 (d, 1H, H-1′, ³J_(1′-F)=17.1Hz), 6.25 (m, 1H, OH-3′), 5.33 (ft, 1H, OH-5′), 4.68 (m, 1H, H-3′),4.00-3.69 (m, 3H, H-4′, H-5′, H-5″), 3.77 (d, 1H, ethynyl, ⁴J_(H-F)=5.4Hz). ¹³C NMR (DMSO-d₆, 75 MHz) δ(ppm) 155.8 (C-4), 152. (C-2), 149.0(C-6), 138.7 (C-8), 118.5 (C-5), 95.4 (d, C-2′, ¹J_(2′-F)=180.8 Hz),87.6 (d, C-1′, ²J_(1′-F)=40.5 Hz), 82.5 (d, CCH, ³J_(C-F)=8.0 Hz), 82.0(C-4′), 74.5 (d, CCH, ²J_(C-F)=31.0 Hz), 72.8 (d, C-3′, ²J_(3′-F)=19.5Hz), 59.2 (C-5′). ¹⁹F NMR (DMSO-d₆, 235 MHz) δ(ppm) −158.0 (t). LC/MS:(M+H⁺) 294.1 (5.74 min). UV λ_(max) 258 nm. R_(f) 0.33 (MeOH/CH₂Cl,15/85, v/v).

4b:N²-Isobutyryl-9-[3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethylsilylethynyl-β-D-arabino-furanosyl]guanine

To a suspension of CrO₃ (110.76 mmol) in DCM (220 mL) at 0° C., aceticanhydride (110.76 mmol) and anhydrous pyridine (221.52 mmol) were added.9-[3,5-O-(1,3-Diyl-1,1,3,3-tetraisopropyldisiloxane)-ribo-furanosyl]-N²-isobutyrylguanine(36.92 mmol) in solution in DCM (110 mL) was added dropwise. The coolingbath was removed and the resulting solution stirred at room temperaturefor 5 h. The reaction mixture was poured into cold ethyl acetate,filtered through a silica and celite gel plug, concentrated to drynessand coevaporated twice with toluene. The residue obtained was dissolvedin DCM and stirred with an excess of MgSO₄ overnight, filtered andevaporated to get the ketone. The trimethylsilylacetylene (88.60 mmol)was dissolved in anhydrous THF (98 mL) under argon. n-Butyllithium (55.4mL, 1.6 M in hexanes) was added dropwise at −78° C. The reaction mixturewas stirred for 30 min at −78° C. and then allowed to warm up to −55° C.The ketone in solution in THF (49 mL) was added dropwise at −78° C. Thereaction mixture was stirred for 1 h at −78° C. and then allowed to warmup to −30° C. and stirred for 3 h. The reaction was quenched by carefuladdition of aqueous saturated NH₄Cl (72 mL) at −78° C. After warming toroom temperature, the mixture was diluted with ethyl acetate, washedtwice with saturated brine, dried (Na₂SO₄) and concentrated to dryness.The crude material was purified using column chromatography eluting with1.5% MeOH in dichloromethane to give the title compound (8.59 g, 34%, 2steps). Pale yellow foam. Molecular Formula C₃₁H₅₅N₅O₆Si₃. ¹H NMR(DMSO-d₆, 250 MHz) δ(ppm) 12.10 (ls, 1H, NH), 11.69 (ls, 1H, NH), 7.91(s, 1H, H-8), 6.69 (s, 1H, OH), 5.94 (s, 1H, H-1′), 4.29 (d, 1H, H-3′,J_(3′-4′)=5.5 Hz), 3.85-3.95 (m, 3H, H-4′, H-5′ and H-5″), 2.46 (m, 1H,CH(CH₃)₂), 0.90-1.08 (m, 30H, iPr and CH(CH₃)₃), 0.00 (s, 9H, Si(CH₃)₂).LC/MS: (M+H⁺) 692.4 (24.96 min). UV λ_(max1) 254 nm, λ_(max2) 281 nm.R_(f) 0.34 (MeOH/CH₂Cl, 15/85, v/v).

5b:N²-Isobutyryl-9-[(2R)-2-deoxy-2-fluoro-3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethyl-silylethynyl-β-D-erythro-pentofuranosyl]guanine

4b (2.89 mmol) was dissolved in dried DCM (60 mL) under argon andpyridine (18.06 mmol) was added. The reaction mixture was cooled to −20°C. and DAST (31.35 mmol) was added dropwise. The cooling bath wasremoved after completion of the addition. Stirring was continued for 1 h15 and the mixture was dissolved with ethyl acetate and poured intosaturated NaHCO₃ and stirred for 5 min. The organic layer was washedwith saturated brine, dried (Na₂SO₄), concentrated, and purified bysilica gel chromatography eluting with ethyl acetate in DCM (2%) to givethe title compound (1.41 g, 70%). Yellow oil. Molecular FormulaC₃₁H₅₄FN₅O₅Si₃. ¹H NMR (DMSO-d₆, 250 MHz) δ (ppm) 12.22 (s, 1H, NH),8.09 (s, 1H, H-8), 6.21 (d, 1H, H-1′, J_(1′-F)=15.6 Hz), 4.54 (dd, 1H,H-3′, J_(3′-F)=23.6 Hz, J_(3′-4′)=9.8 Hz), 4.33 (m, 1H, H-5′,²J_(5′-5″)=13.1 Hz), 4.16 (m, 1H, H-5″), 2.81 (m, 1H, CH(CH₃)₂),1.13-1.03 (m, 34H, iPr and CH(CH₃)₂), 0.08 (s, 9H, Si(CH₃)₃,³J_(H-H)=6.9 Hz). ¹⁹F NMR (DMSO-d₆, 235 MHz) δ(ppm) −160.26 (dd,J_(f-1′)=16.1 Hz, J_(F-3′)=23.3 Hz). LC/MS: (M+H⁺) 694.7 (24.02 min).LRFAB-MS (GT): 694 (M−H)⁺, 692 (M−H)⁻. UV λ_(max) 256 nm. R_(f) 0.46(MeOH/CH₂Cl, 05/95, v/v).

6b:N²-Isobutyryl-9-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]guanine

5b (1.89 mmol) was dissolved in methanol (13.8 mL) and ammonium fluoride(24.54 mmol) was added. The resulting solution was stirred at reflux for1 h and evaporated to dryness. The crude material was purified on silicagel chromatography eluting with a stepwise gradient 6-10% of methanol inDCM to yield the title compound (344 mg, 48%). Pale yellow oil.Molecular Formula C₁₆H₂₀FN₅O₄Si₃. ¹H NMR (DMSO-d₆, 400 MHz) δ(ppm) 12.18(ls, 1H, NH), 11.77 (ls, 1H, NH), 8.34 (s, 1H, H-8), 6.29 (d, 1H, OH-3′,J_(OH-3′)=7.5 Hz), 6.20 (d, 1H, H-1′, J_(1′-F)=16.2 Hz), 5.39 (t, 1H,OH-5′, J_(OH-5′)=5.1 Hz), 4.52 (dt, 1H, H-3′, J_(3′-F)=22.9 Hz), 3.98(m, 1H, H-4′), 3.90-3.85 (m, 2H, H-5′ and ethynyl), 3.72 (m, 1H, H-5″),2.52 (m, 1H, CH(CH₃)₂), 1.14 (d, 6H, CH(CH₃)₂, ³J_(H-H)=6.9 Hz). ¹³C NMR(DMSO-d₆, 100 MHz) δ(ppm) 180.7 (C-6), 155.3 (C-2), 148.9 (C-4), 137.3(C-8), 120.4 (C-5), 95.8 (d, C-2′, ¹J_(2′-F)=182.1 Hz), 87.7 (d, C-1′,²J_(1′-F)=39.2 Hz), 83.4 (d, CCH, ³J_(C-F)=9.1 Hz), 82.6 (C-4′), 75.9(d, CCH, ²J_(C-F)=31.2 Hz), 72.9 (d, C-3′, ²J_(3′-F)=19.1 Hz), 59.3(C-5′). ¹⁹F NMR (DMSO-d₆, 235 MHz) δ(ppm) −158.9 (m). LC/MS: (M+H⁺)380.3 (8.34 min). UV λ_(max1) 256 nm, R_(f) 0.40 (MeOH/CH₂Cl, 15/85,v/v).

9-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]guanine

6b (1.33 mmol) was dissolved in saturated methanolic ammonia (62 mL) andstirred at room temperature for 20 h. The reaction mixture was thenevaporated to dryness under reduced pressure. The residue was dissolvedin water and washed twice with ethyl acetate. The aqueous layer wasevaporated and purified on reverse phase column chromatography (C 18)eluting with a gradient 2-15% of acetonitrile in water. The residueobtained was then dissolved in hot ethyl acetate, filtered and dried togive the title compound (134 mg, 33%). Yellow solid. Molecular FormulaC₁₂H₁₂FN₅O₄ ¹H NMR (DMSO-d₆, 400 MHz) δ(ppm) 10.70 (ls, 1H, NH), 7.98(s, 1H, H-8), 6.60 (ls, 2H, NH₂), 6.21 (d, 1H, OH-3′, J_(OH-3′)=7.6 Hz),5.83 (d, 1H, H-1′, J_(1′-F)=16.9 Hz), 5.29 (t, 1H, OH-5′, J_(OH-5′)=5.2Hz), 4.50 (td, 1H, H-3′, J_(3′-F)=22.8 Hz, J_(3′-4′)=9.2 Hz), 3.93-3.81(m, 3H, H-4′, H-5′ and ethynyl), 3.70 (m, 1H, H-5″). ¹³C NMR (DMSO-d₆,100 MHz) δ(ppm) 157.2 (C-6), 154.3 (C-2), 151.05 (C-4), 135.1 (C-8),116.7 (C-5), 96.4 (d, C-2′, ¹J_(C-F)=182.1 Hz), 87.4 (d, C-1′,²J_(C-F)=39.2 Hz), 83.1 (d, CCH, J_(C-F)=9.1 Hz), 82.4 (C-4′), 76.2 (d,CCH, ²J_(C-F)=31.2 Hz), 73.2 (d, C-3′, ²J_(C-F)=20.1 Hz), 59.5 (C-5′).¹⁹F NMR (DMSO-d₆, 235 MHz) δ(ppm) −158.5 (m). LC/MS (A): (M+H⁺) 310.1(5.55 min). LRFAB-MS (GT): 619 (2M-41)⁺, 310 (M+H)⁺, 152 (B+H)⁺, 617(2M−H)⁻, 308 (M−H)⁻. UV λ_(max) 253 nm

3c:1-[2-oxo-3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-β-D-ribo-furanosyl]uracile

3c was synthesized from1-[3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-ribo-furanosyl]uracileas described for 3a. Pale yellow foam. Molecular Formula C₂₁H₃₆N₂O₇Si₂¹H NMR (DMSO-d₆, 400 MHz) δ(ppm) 11.58 (ls, 1H, NH), 7.74 (d, 1H, H-6,J₆₋₅=8.0 Hz), 5.68 (d, 1H, H-5, J₅₋₆=8.0 Hz), 5.45 (s, 1H, H-1′), 4.97(d, 1H, H-3′, J_(3′-4′)=9.2 Hz), 4.06-3.90 (m, 3H, H-4′, H-5′),1.14-0.87 (m, 28H, iPr). LR LC/MS: (M+H⁺) 485.1 (M−H⁻) 483.1 (5.53 min).UV λ_(max) 262 nm. R_(f) 0.40 (MeOH/CH₂Cl, 05/95, v/v).

4c:1-[3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethylsilylethynyl-β-D-arabino-furanosyl]uracileYoshimura, Y.; Iino, T.; Matsuda, A. Tetrahedron Lett. 1991, 32,6003-6006.

Molecular Formula C₂₆H₄₆N₂O₇Si₃ ¹H NMR (DMSO-d₆, 400 MHz) δ(ppm) 11.35(ls, 1H, NH), 7.44 (d, 1H, H-6, J₆₋₅=8.0 Hz), 6.54 (s, 1H, OH), 6.02 (s,1H, H-1′), 5.54 (d, 1H, H-5, J₆₋₅=8.0 Hz), 4.13-3.93 (m, 3H, H-3′,H-5′), 3.75 (m, 1H, H-4′), 1.03-0.96 (m, 28H, iPr), 0.00 (s, 9H,Si(CH3)3). ¹³C NMR (DMSO-d₆, 100 MHz) δ(ppm) 163.4 (C-4), 150.8 (C-2),141.6 (C-6), 103.6 (CCSi), 101.2 (C-5), 92.5 (CCSi), 87.3 (C-1′), 80.9(C-4′), 77.9 (C-2′), 75.9 (C-3′), 61.8 (C-5′), 17.7-17.1 (8C, 4SiC(CH₃)₂), 13.3-12.6 (4C, 4 SiC(CH₃)₂), 0.2 (3C, Si(CH₃)₃). LR LC/MS:(M+H⁺) 583.2 (M−H⁻) 581.2 (6.72 min). UV λ_(max) 261 nm. R_(f) 0.27(Ethyl acetate/CH₂Cl, 10/90, v/v).

5c:1-[(2R)-2-Deoxy-2-fluoro-3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethylsilylethynyl-β-D-erythro-pentofuranosyl]uracile

5c was synthesized from 4c as described for 5a. Yellow oil. MolecularFormula C₂₇H₄₉FN₂O₆Si₃ ¹H NMR (DMSO-d₆, 400 MHz) δ(ppm) 11.62 (sl, 1H,NH), 7.43 (dl, 1H, H-6, J₆₋₅=8.0 Hz), 6.12 (d, 1H, H-1′, J_(1′-F)=16.8Hz), 5.68 (d, 1H, H-5, J₅₋₆=8.0 Hz), 4.22-3.85 (m, 4H, H-3′, H-4′,H-5′), 1.16-1.00 (m, 28H, iPr), 0.00 (s, 9H, Si(CH₃)₃). ¹⁹F NMR(DMSO-d₆, 376 MHz) δ(ppm) −159.7. LR LC/MS: (M+H⁺) 585.2 (M−H⁻) 583.3(6.47 min). UV λ_(max) 261 nm. R_(f) 0.52 (Ethyl acetate/CH₂Cl, 15/85,v/v).

6c:1-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]uracile

A mixture of 5c (0.56 mmol) and ammonium fluoride (7.31 mmol) weredissolved in methanol (10 mL) stirred at reflux for 1 h and evaporatedto dryness. The resulting residue was purified on silica gel flashcolumn chromatography eluting with a gradient 0-20% methanol in DCM andthen, on reverse phase column chromatography eluting with a gradient0-100% acetonitrile in water to give the desired product which waslyophilised from water (47 mg, 31%). White lyophilised powder. MolecularFormula C₁₁H₁₁FN₂O₅. ¹H NMR (DMSO-d₆, 400 MHz) δ(ppm) 11.49 (sl, 1H,NH), 7.87 (d, 1H, H-6, J₆₋₅=8.0 Hz), 6.18 (d, 1H, OH-3′, J_(OH-3′)=7.2Hz), 6.10 (d, 1H, H-1′, J_(1′-F)=18.0 Hz), 5.69 (d, 1H, H-5, J₅₋₆=8.0Hz), 5.32 (m, 1H, OH-5′), 4.19-4.10 (m, 2H, H-3′ and ethynyl), 3.85-3.75(m, 2H, H-4′ H-5′), 3.60 (m, 1H, H-5″). ¹³C NMR (DMSO-d₆, 100 MHz)δ(ppm) 163.3 (C-4), 150.6 (C-2), 140.1 (C-6), 102.5 (C-5), 95.5 (d,C-2′, J_(2′-F)=186.1 Hz), 87.1 (d, C-1′, ²J_(1′-F)=40.2 Hz), 83.2 (d,CCH, ²J_(C-F)=8.0 Hz), 82.1 (C-4′), 76.5 (d, CCH, ⁴J_(C-F)=30.1 Hz),73.3 (d, C-3′, ²J_(C-F)=19.1 Hz), 58.7 (C-5′). ¹⁹F NMR (DMSO-d₆, 376MHz) δ(ppm) −158.2. LR LC/MS: (M+H⁺) 271.1 (M−H⁻) 269.2 (1.12 min).HRFAB-MS C₁₁H₁₂O₅N₂F. (M+H⁺) calculated 271.0730. found 271.0739. UVλ_(max) 261 nm. R_(f) 0.33 (MeOH/CH₂Cl, 20/80, v/v).

2d:1-[3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-β-D-ribo-furanosyl]thymine

The 1-(β-D-ribo-furanosyl)thymine (40.9 mmol) was dissolved in pyridine(435 ml) and the mixture was cooled down to 0° C. with an ice-bath for25 minutes. Then, TIPSCl₂ (16.2 ml) was added and after completeaddition, the mixture was allowed to warm up to room temperature. Thereaction mixture was stirred at room temperature for 3 hrs, diluted withdichloromethane and water, washed with a saturated aqueous solution ofNaHCO₃. The organic phases were combined, dried over Na₂SO₄, filteredand evaporated. The residue was coevaporated with toluene to removepyridine. The resulting residue was purified by flash columnchromatography eluting with a gradient 0-2% of methanol indichloromethane to give the title compound. Off-white powder. MolecularFormula C₂₂H₄₀N₂O₇Si₂, ¹H NMR (DMSO-d₆, 400 MHz) δ(ppm) 0.94-1.04 (m,28H), 1.73 (s, 3H), 3.86-3.96 (m, 1H), 4.06-4.13 (m, 2H), 4.14-4.20 (m,1H), 5.44-5.48 (m, 1H), 5.53 (brs, 1H), 5.77 (brs, 1H) 7.42 (s, 1H),11.35 (brs, 1H). UV λ_(max) 212 nm, 266 nm.

3d:1-[2-oxo-3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-β-D-ribo-furanosyl]thymine

To a suspension of CrO₃ (60 mmol) in dichloromethane (200 mL) at 0° C.,acetic anhydride (59 mmol) and anhydrous pyridine (120 mmol) were added.2d (20 mmol) in solution in DCM was added dropwise. The cooling bath wasremoved and the resulting solution stirred at room temperature for 3 h.The reaction mixture was poured into cold ethyl acetate, filteredthrough a silica and celite gel plug, concentrated to dryness andcoevaporated twice with toluene to give the title compound. Colorlessoil. Molecular Formula C₂₂H₃₈N₂O₇Si₂

4d:1-[3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethylsilylethynyl-β-D-arabino-furanosyl]thymine

4d was synthesized from 3d and trimethylacetylene as described for 4a.Brown solid. Molecular Formula C₂₇H₄₈N₂O₇Si₃. Scan ES⁺ 597 (M+H)⁺, UVλ_(max) 265 nm.

5d:1-[(2R)-2-Deoxy-2-fluoro-3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethyl-silylethynyl-β-D-erythro-pentofuranosyl]thymine

5d was synthesized from 4a as described for 5a. Brown solid. MolecularFormula C₂₇H₄₇FN₂O₆Si₃ ¹H NMR (CDCl₃-d₆, 400 MHz) δ(ppm) 0.1 (s, 9H),1.05-1.14 (m, 28H), 1.92 (s, 3H), 3.99-4.13 (m, 1H), 4.44-4.9 (m, 3H),6.35 (d, 1H, J=16.44 Hz), 7.2 (s, 1H), 8.86 (s, 1H). Scan ES⁺ 599(M+H)⁺,

6d:1-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]thymine

6d was synthesized from 5d as described for 7i. Molecular FormulaC₁₂H₃FN₂O₅. ¹H NMR (CDCl₃-d₆, 400 MHz) δ(ppm) 1.75 (s, 3H), 3.6-3.65 (m,1H), 3.82-3.84 (m, 2H), 4.07 (d, 1H, J=5.27 Hz), 4.19 (m, 1H), 5.4 (brs,1H), 6.08 (d, 1H, J=17.8 Hz), 6.17 (brs, 1H), 7.8 (s, 1H), 11.46 (brs,1H). Scan ES⁺ 285 (M+H)⁺, UV λ_(max) 266 nm.

4e:N⁴-Benzoyl-1-[3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethylsilylethynyl-β-D-arabino-furanosyl]cytosine

4e was synthesized from 3e and trimethylacetylene as described for 4a.Brown solid. Molecular Formula C₃₃H₅₁N₃O₇Si₃ Scan ES⁺ 686 (M+H)⁺, UVλ_(max) 260 nm, 310 nm.

5e:N⁴-benzoyl-1-[(2R)-2-Deoxy-2-fluoro-3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethyl-silylethynyl-β-D-erythro-pentofuranosyl]cytosine

5e was synthesized fromN⁴-benzoyl-1-[3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethyl-silylethynyl-D-arabino-furanosyl]cytosineas described for 5a. Yellow solid. Molecular Formula C₃₀H₄₂FN₃O₆Si₂.Scan ES⁺ 688 (M+H)⁺, UV λ_(max) 260 nm, 310 nm.

6e:N⁴-benzoyl-1-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]cytosine

6e was synthesized from 5e as described for 7i. White powder. MolecularFormula C₁₈H₁₆FN₃O₅. ¹NMR (DMSO-d₆, 400 MHz) δ(ppm) 3.63-3.69 (m, 1H),3.82-0.393 (m, 2H), 4 (d, 1H, J=5.27 Hz), 4.13-4.24 (m, 1H), 5.38 (brs,1H), 6.23-6.28 (m, 2H), 7.32-7.36 (m, 1H), 7.49-7.53 (m, 2H), 7.6-7.64(m, 1H), 7.99-8.01 (m, 2H), 8.34 (d, 1H, J=7.32 Hz), 11.30 (brs, 1H).Scan ES⁺ 374 (M+H)⁺, UV λ_(max) 262 nm, 303 nm.

7k:1-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]cytosine

Molecular Formula C₁₁H₁₂FN₃O₄. ¹H NMR (DMSO-d₆, 400 MHz) δ(ppm)3.57-3.62 (m, 1H), 3.77-3.80 (m, 2H), 3.95 (d, 1H, J=5.53 Hz), 4.03-4.16(m, 1H), 5.2 (brs, 1H), 5.73 (d, 1H, J=7.19 Hz), 6.06 (d, 1H, J=7.19Hz), 6.14-6.25 (m, 1H), 7.17-7.3 (2 brs, 2H), 7.74 (d, 1H, J=7.74 Hz).Scan ES⁺ 270 (M+H)⁺, UV λ_(max) 271 nm.

8k:N⁴-dimethoxytrityl-1-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]cytosine

To a stirred solution of 7k (2.34 mmol) in pyridine (7.2 ml) was addedtrimethylsilyl chloride (9.36 mmol) at room temperature. The reactionmixture was stirred at room temperature for 2 hours. 4-dimethylaminopyridine (1.17 mmol) and dimethoxytrityl chloride (3.51 mmol) were thenadded. The mixture was stirred at room temperature for 16 hours. Thereaction mixture was diluted with DCM and sat NaHCO₃ solution. Theorganic phase was washed twice with sat NaHCO₃ solution, dried overNa₂SO₄, filtered and evaporated. The crude material was dissolved in asolution NH4OH/dioxin (2:1) and stirred for 4 hrs. Solvent wasevaporated and the residue purified by silica gel chromatography(DCM/EtOH) to yield the title compound. White foam. Molecular FormulaC₃₂H₃₀FN₃O₆. Scan ES⁻ 570 (M+H)⁻, UV λ_(max) 277 nm

9k:1-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]-4-N-dimethoxytrityl-cytosin-5′-yl-bis(S-pivaloyl-2-thioethylphosphate

To a stirred solution of 8k (0.35 mmol) in and THF/tetrazole solution(1.05 mmol) was addedbis(S-pivaloyl-2-thioethyl)N,N-diisopropylphosphoramidite (0.42 mmol) at0° C. The reaction mixture was stirred at room temperature for 3 hrs.The reaction mixture was cooled down to 0° C. and tert-butylhydroperoxide (0.7 ml/mmol) was added. The reaction mixture was stirredat room temperature for 2 hours. The mixture was diluted with DCM,neutralized with sat Na₂S₂O₃ solution. The organic phase was washedtwice with H₂O, extracted, dried over Na₂SO₄, filtered and evaporated.The crude material was purified by silica gel chromatography (DCM/EtOH)to yield the title compound. Glassy compound. Molecular FormulaC₄₇H₅₉FN₃O₁₁PS₂. Scan ES⁺ 938 (M+H)⁺, UV λ_(max) 277 nm

11k:1-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]-cytosin-5′-yl-bis(S-pivaloyl-2-thioethylphosphate

9k (34 mmol) was stirred in AcOH/MeOH/H₂O (3/6/1) solution for 2 hrs andat 50° C. for 4 hours. The reaction mixture was then evaporated andpurified by silica gel chromatography (DCM/EtOH) to yield the titlecompound. White lyophilized powder. Molecular Formula C₂₅H₃₇FN₃O₉PS₂. ¹HNMR (DMSO-d₆, 400 MHz) δ (ppm) 1.17 (s, 18H), 3.08-3.11 (t, J=6.07 Hz,4H), 3.99-4.08 (m, 7H), 4.22-4.28 (m, 2H), 5.73-5.75 (d, J=7.30 Hz, 1H),6.30 (brs, 2H), 7.26-7.31 (d, J=17.30 Hz, 2H), 7.47-7.48 (d, J=7.30 Hz,1H) ¹⁹F NMR (DMSO-d₆, 376 MHz) δ (ppm) −156.48 (s, 1F) ³¹P NMR (DMSO-d₆,162 MHz) δ (ppm) −1.96 (s, 1P). Scan ES⁺ 638 (M+H)⁺, UV λ_(max) 271 nm

12:N²-Methoxytrityl-9-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]guanine

To a stirred solution of 7i (5.18 mmol) in pyridine (7 ml/mmol) wasadded trimethylsilyl chloride at room temperature. The mixture wasstirred at room temperature for 6 hours. Methoxytrityl chloride (6.21mmol) was then added and the reaction mixture was stirred at roomtemperature for 16 hours and 2 hours with NH₄OH (4 ml/mmol). The mixturewas diluted with ethyl acetate, washed with H₂O, sat NaHCO₃ solution andsat NaCl solution, dried over Na₂SO₄, filtered and evaporated. The crudematerial was purified by silica gel chromatography (DCM/MeOH) to yieldthe title compound. Yellowish oil. Molecular Formula C₃₂H₂₈FN₅O₅.

13:N²-Methoxytrityl-9-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-5-O-tert-butyldimethylsilyl-β-D-erythro-pentofuranosyl]guanine

To a stirred solution of 12 (2.29 mmol) in pyridine (5 ml) at 0° C., wasadded tert-butyldimethylsilyl chloride (2.75 mmol). The reaction mixturewas stirred at room temperature for 24 hours. It was then diluted in DCMand washed twice with H₂O. The organic phase was extracted, dried overNa₂SO₄, filtered and evaporated. The crude material was purified bysilica gel chromatography (DCM/MeOH) to yield the title compound.Yellowish oil. Molecular Formula C₃₈H₄₄FN₅O₅Si. Scan ES⁺ 696 (M+H)⁺,λ_(max) 260 nm. Scan ES⁻ 694 (M+H)⁻, UV λ_(max) 260 nm

14:N²-Methoxytrityl-9-[(2R)-2,3-dideoxy-2-C-ethynyl-2-fluoro-5-O-tert-butyldimethylsilyl-β-D-g/ycero-pentofuranosyl]guanine

To a stirred solution of 13 (0.14 mmol) in acetonitrile (47 ml/mmol) wasadded 4-dimethylamino pyridine (0.56 mmol) and phenylchlorothionoformate (0.43 mmol) at room temperature. The reactionmixture was stirred at room temperature for 16 hours and wasconcentrated under reduced pressure. The residue obtained was dissolvedin DCM, the organic phase was washed with H₂O, HCl (1N), dried overNa₂SO₄, filtered evaporated and co-evaporated with toluene.

The crude material was dissolved in toluene (12 ml/mmol),azo-bis-isobutyronitrile (0.02 mmol) and tributylstannane (0.24 mmol)were added at room temperature. The reaction mixture was stirred at 125°C. for 2 hours and concentrated under reduced pressure. The crudematerial was purified by silica gel chromatography (DMC/MeOH) to yieldthe title compound. Yellowish oil. Molecular Formula C₃₈H₄₄FN₅O₄Si. ScanES⁺ 680 (M+H)⁺, UV λ_(max) 260 nm

15:N²-Methoxytrityl-9-[(2R)-2,3-dideoxy-2-C-ethynyl-2-fluoro-β-D-glycero-pentofuranosyl]guanine

14 (0.35 mmol) was dissolved in MeOH (20 ml/mmol). Ammonium fluoride(3.55 mmol) was then added at room temperature and the reaction mixturewas stirred at 70° C. for 2 hours. After concentration under reducedpressure, the crude material was purified by silica gel chromatography(DCM/MeOH) to yield the title compound. Beige foam. Molecular FormulaC₃₂H₃₀FN₅O₄. ¹H NMR (CDCl₃-d₆, 400 MHz) δ (ppm) 2.38-2.45 (m, 2H), 2.75(brs, 2H), 3.64-3.67 (d, J=12.20 Hz, 2H), 3.77 (s, 4H), 4.20-4.23 (d,J=11.7 Hz, 1H), 4.41-4.42 (d, J=8.4 Hz, 1H), 5.83-5.87 (d, J=16.24 Hz,1H), 6.80-6.82 (d, J=8.12 Hz, 4H), 7.26-7.31 (m, 11H), 7.84 (brs, 1H),9.26 (brs, 1H)

16:9-[(2R)-2,3-Dideoxy-2-C-ethynyl-2-fluoro-βD-glycero-pentofuranosyl]guanine

15 (0.09 mmol) was stirred in AcOH/THF/H₂O (3/6/1) solution at 50° C.for 1 day. The reaction mixture was then concentrated under reducedpressure and purified by silica gel chromatography, C18 (H₂O/ACN) Beigelyophilisated powder. Molecular Formula C₁₂H₁₂FN₅O₅. ¹H NMR (DMSO-d₆,400 MHz) δ (ppm) 2.57-2.74 (m, 2H), 3.56 (s, 1H), 3.61-3.64 (d, J=12.10Hz, 1H), 3.79-3.82 (d, J=12.10 Hz, 1H), 3.91-3.93 (d, J=5.40 Hz, 1H),4.32-4.35 (m, 1H), 5.25 (s, 1H), 6.06-6.10 (d, J=18.20 Hz, 1H), 6.64 (s,1H), 8.01 (s, 1H), 10.82 (s, 1H) ¹⁹F NMR (DMSO-d₆, 376 MHz) δ (ppm)−138.4 (s, 1F). Scan ES 292 (M+H)⁻, Scan ES⁺ 316 (M+Na)⁺, UV λ_(max) 251nm

17:9-[(2R)-2,3-Dideoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]-guanin-5′-yl-bis(S-pivaloyl-2-thioethylphosphate)

17 was synthesized from 14 (0.35 mmol) as described for 9. The crudematerial was then stirred in AcOH/THF/H₂O (4/2/1) at 50° C., for 3hours. The reaction mixture was concentrated under reduced pressure andpurified by silica gel chromatography (DCM/MeOH) to yield the titlecompound. Beige solid. Molecular Formula C₂₆H₃₇FN₅O₈PSi₂ ¹H NMR(DMSO-d₆, 400 MHz) δ (ppm) 2.48 (s, 18H), 2.65-2.68 (m, 2H), 3.06-3.10(q, J=3.71 Hz, and J=6.02 Hz, 4H), 3.97-4.04 (m, 5H), 4.31-4.35 (m, 2H),4.50-4.52 (m, 1H), 6.13-6.18 (d, J=17.60 Hz, 1H), 6.63 (s, 2H), 7.82 (s,1H), 10.85 (s, 1H). ¹⁹F NMR (DMSO-d₆, 376 MHz) δ (ppm) −139.2 (s, 1F).Scan ES⁺ 662 (M+H)⁺, UV λ_(max) 254 nm. HPLC (0-100% ACN over a periodof 8 min) t_(R)=5.65 min.

18:N²-Methoxytrityl-9-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-5-O-tert-butyldimethylsilyl-3-O-tetrahydropyranyl-β-D-erythro-pentofuranosyl]guanine

To a stirred solution of 13 (0.8 mmol), in and THF (20 ml/mmol), at roomtemperature, was added p-toluen sulfonic acid (0.12 mmol) anddihydropyran (2 ml/mmol). The reaction mixture was stirred at roomtemperature for 3 days and neutralized with TEA. The mixture was dilutedwith DCM, washed twice with H₂O. The organic phase was dried overNa₂SO₄, filtered and evaporated. The crude material was purified bysilica gel chromatography (DCM/MeOH) to yield the title compound.Molecular Formula C₄₃H₅₂FN₅O₆Si. Scan ES⁺ 780 (M+H)⁺

19:N²-Methoxytrityl-9-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-3-O-tetrahydropyranyl-β-D-erythro-pentofuranosyl]guanine

19 was synthesized from 8 described for 15. Molecular FormulaC₃₇H₃₈FN₅O₆. Scan ES⁺ 666 (M+H)⁺.

21:N²-Tetrahydropyranyl-9-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-5-O-tert-butyldimethylsilyl-3-O-tetrahydropyranyl-β-D-erythro-pentofuranosyl]guanine

21 was obtained from the purification of 18. Molecular FormulaC₂₈H₄₂FN₅O₆Si. Scan ES⁺ 592 (M+H)⁺, UV λ_(max) 273 nm

22:N²-Tetrahydropyranyl-9-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-3-O-tetrahydropyranyl-β-D-erythro-pentofuranosyl]guanine

22 was synthesized from 21 (0.46 mmol), as described for 15. MolecularFormula C₂₂H₂₈FN₅O₆

20:9-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]guanin-5′-yl-bis(S-pivaloyl-2-thioethylphosphate

20 was synthesized from 19 (0.09 mmol), as described for 9k. The crudematerial was then stirred at room temperature in AcOH/THF/H₂O (4/2/1)solution overnight. The reaction mixture was concentrated under reducedpressure and purified by silica gel chromatography (DCM/MeOH) to yieldthe title compound. White lyophilized powder. Molecular FormulaC₂₆H₃₇FN₅O₉PS₂ ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 1.16 (s, 20H),3.06-3.09 (m, 4H), 3.90-3.91 (d, J=5.40 Hz, 1H), 3.99-4.10 (q, J=6.70 Hzand J=7.00 Hz, 4H), 4.32-4.38 (m, 2H), 4.63 (m, 1H), 6.10-6.14 (d,J=16.93 Hz, 1H), 6.69 (s, 2H), 7.79 (s, 1H), 10.96 (s, 1H) ³¹P NMR(DMSO-d₆, 162 MHz) δ (ppm) −1.91 (s, 1P) ¹⁹F NMR (DMSO-d₆, 376 MHz) δ(ppm) −156.82 (s, 1F) Scan ES⁺ 678 (M+H)⁺.

25:1-[(2R)-2-Deoxy-2-fluoro-3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethylsilylethynyl-β-D-erythro-pentofuranosyl]-4-thiouracile

5c (820 mg, 1.40 mmol) was dissolved in anhydrous 1,2-dichloroethane (35mL) and treated with Lawesson's reagent (1.13 g, 2.80 mmol). Thereaction mixture was stirred at reflux overnight and evaporated todryness. The resulting residue was filtered on a silica gel plug elutingwith a gradient 0-5% of ethyl acetate in dichloromethane to give thetitle compound. Yellow oil. Molecular Formula C₂₆H₄₅FN₂O₅SSi₃ LR LC/MS:(M+H⁺) 601.3 (M−H⁻) 599.3 (7.03 min). UV λ_(max) 332 nm. R_(f) 0.71(Ethyl acetate/CH₂Cl, 7/93, v/v).

26:1-[(2R)-2-Deoxy-2-fluoro-3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisilosane)-2-C-ethynyl-β-D-erythro-pentofuranosyl]cytosine

Crude 25 was dissolved in saturated ammoniacal methanol (9 mL). Theresulting solution was heated by micro-waves at 120° C. for 20 min andconcentrated under reduced pressure to give the title compound. Oilyresidue. Molecular Formula C₂₆H₄₆FN₃O₅Si₃ LR LC/MS (B): (M+H⁺) 512.3(M−H⁻) 510.3 (5.33 min). UV λ_(max1) 242 nm, λ_(max2) 273 nm.

27i:9-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]adenine5′-triphosphate sodium salt

To a solution of 7i (0.286 mmol) in triethylphosphate (750 phosphorylechloride (75 μL, 0.807 mmol) was added at 0° C. This reaction mixture Awas stirred overnight at 5° C. Tributylammonium pyrophosphate (PPi/Bu₃N1/1.5, 1 g, 2.19 mmol) was dissolved in anhydrous DMF (2 mL).Tributylamine (420 μL, 1.76 mmol) was added to the PPi and the resultingmixture was stirred for 15 min at 0° C. 2.4 mL of this solution wereadded to the reaction mixture A. The reaction mixture was stirred at 0°C. for 1 min. The reaction was carefully quenched with TEAB 1M (pH=7.5,10 mL), stirred 20 min at 0° C., then diluted with water and ethylacetate. The aqueous phase was concentrated under reduced pressure. Thecrude material was subjected to DEAE-Sephadex chromatography elutingwith a gradient 10⁻³-1 M of TEAB). The desired fractions were combined,concentrated under reduced pressure and coevaporated with a mixture ofwater/methanol, and finally coevaporated with water. The resultingresidue was purified on semipreparative HPLC. Fractions containing theexpected product were concentrated under reduced pressure, coevaporatedwith a mixture of water/methanol and lyophilised from water. Thetriethylammonium salt triphosphate was eluted three times with water ona Dowex Na⁺ resin column to yield after lyophilisation from water to thesodium salt.

Molecular Formula C₁₂H₁₁FN₅O₁₂P₃ 3Na. ¹H NMR (D₂O, 300 MHz) δ(ppm) 8.31(s, 1H, H-8), 8.14 (s, 1H, H-2), 6.28 (d, 1H, H-1′, ³J_(1′-F)=15.6 Hz),4.64 (m, 1H, H-3′), 4.42 (m, 1H, H-5′), 4.35-4.25 (m, 2H, H-4′ andH-5″), 2.82 (d, 1H, ethynyl, ⁴J_(H-F)=5.5 Hz). ³¹P NMR (D₂O, 121 MHz)δ(ppm) −10.27 (d, 1P, P_(γ), J_(Pγ-Pβ)=19.4 Hz), −11.03 (d, 1P, P_(α),J_(Pα-Pβ)=19.4 Hz), −22.38 (t, 1P, P_(β), J_(Pβ-Pγ)=J_(Pβ-Pα)=19.4 Hz).¹⁹F NMR (D₂O, 282 MHz) δ(ppm) −160.0 (m). LRFAB-MS (GT): 600 (M+H)⁺, 578(M−Na+2H)⁺, 556 (M−2Na+3H), 598 (M−H)⁻, 576 (M−Na)⁻, 554 (M−2Na⁺H)⁻, 532(M−3Na+2H)⁻.

27j:9-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]guanine5′-triphosphate sodium salt

27j was synthesized from 7j, as described for 27. Molecular FormulaC₁₂H₁₁FN₅O₁₃P₃ 3Na. ¹H NMR (D₂O, 400 MHz) δ(ppm) 7.97 (s, 1H, H-8), 6.19(d, 1H, H-1′, ³J_(1′-F)=16.0 Hz), 4.70 (m, 1H under H₂O, H-3′), 4.39 (m,1H, H-5′), 4.29-4.22 (m, 2H, H-4′ and H-5″), 2.98 (d, 1H, ethynyl,⁴J_(H-F)=5.0 Hz). ³¹P NMR (D₂O, 162 MHz): −10.50 (d, 1P, P_(γ),J_(Pγ-Pβ)=19.4 Hz), −11.03 (d, 1P, P_(α), J_(Pα-Pβ)=19.4 Hz), −22.38 (t,1P, P_(β), J_(Pβ-Pγ)=J_(pβ-Pα)=19.4 Hz). ¹⁹F NMR (DMSO-d₆, 376 MHz)δ(ppm) −159.1 (m). LRFAB-MS (GT): 638 (M+Na)⁺, 616 (M+H)⁺, 594(M−Na+2H)⁺, 572 (M−2Na+3H)⁺, 550 (M−3Na+4H)⁺, 592 (M−Na)⁻, 570(M−2Na+H)⁻, 548 (M−3Na+2H)⁻.

2g:4-Chloro-7-[3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-β-D-ribo-furanosyl]pyrrolo[2,3-d]pyrimidine

2g was synthesized from 9-[β-D-ribo-furanosyl]-7-deaza-6-chloropurine,as described for intermediate 12 Yellow oil. Molecular FormulaC₂₃H₃₅ClN₃O₅Si₂. ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 0.96-1.04 (m, 28H),3.92-3.95 (m, 3H), 4.41-4.58 (m, 2H), 5.65 (s, 1H), 6.08 (s, 1H), 6.71(s, 1H), 7.83 (s, 1H), 8.62 (s, 1H)

3g:4-Chloro-7-[2-oxo-3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-β-D-erythro-pentofuranosyl]pyrrolo[2,3-d]pyrimidine

3g was synthesized from ag as described for 3d. Brown solid. MolecularFormula C₂₃H₃₆ClN₃O₅Si₂. Scan ES⁺ (M+H)⁺ 528, UV λ_(max) 271 nm

4-Chloro-[3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethylsilylethynyl-β-D-arabino-furanosyl]pyrrolo[2,3-d]pyrimidine

4g was synthesized from 3g as described for 4a. Beige solid. MolecularFormula: C₂₈H₄₆ClN₃O₆Si₃. ¹H NMR (DMSO-d₆, 400 MHz) δ(ppm), 0.12 (s,9H), 0.95-1.09 (m, 28H), 3.90-3.94 (m, 1H), 4.02-4.03 (m, 2H), 4.37-4.39(d, J=6.74 Hz, 1H), 6.43 (s, 1H), 6.44 (s, 1H), 6.68 (d, J=3.71 Hz, 1H),7.71-7.72 (d, J=3.84 Hz, 1H), 8.66 (s, 1H)

5g:4-Chloro-7-[(2R)-2-deoxy-2-fluoro-3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethylsilylethynyl-β-D-erythro-pentofuranosyl]pyrrolo[2,3-d]pyrimidine

5g was synthesized from 4g as described for 5a. Yellow oil. Molecularformula C₂₈H₄₆ClFN₃O₆Si_(3.1)H NMR (DMSO-d₆, 400 MHz) δ(ppm) 0.33 (s,9H), 1.02-1.13 (m, 28H), 4.0-4.03 (d, J=13.42 Hz, 1H), 4.12-4.14 (d,J=9.43 Hz, 1H), 4.27-4.31 (d, J=14.00 Hz, 1H), 4.71 (brs, 1H), 6.58-6.62(d, J=17.07 Hz, 1H), 6.82-6.83 (d, J=3.80 Hz, 1H), 7.72 (d, J=3.80 Hz,1H), 8.69 (s, 1H) ¹⁹F NMR (DMSO-d₆, 235 MHz) δ (ppm) −159.6 (s, 1F)

6g:4-Chloro-7-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]pyrrolo[2,3-d]pyrimidine

6g was synthesized from 5g as described for 6a. Yellow oil. MolecularFormula C₁₃H₁₁ClFN₃O₃. ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 3.60-3.65 (d,J=5.44 Hz, 1H), 3.68-3.71 (d, J=12.35 Hz, 1H), 3.85-3.88 (d, J=12.35 Hz,1H), 3.95-3.97 (d, J=8.90 Hz, 1H), 4.46-4.54 (dd, J=23.23 Hz and J=9.39Hz, 1H), 5.38 (s, 1H), 6.28 (s, 1H), 6.57-6.61 (d, J=16.47 Hz, 1H), 6.79(d, J=3.82 Hz, 1H), 8.04 (d, J=3.78 Hz, 1H), 8.70 (s, 1H) ¹⁹F NMR(DMSO-d₆, 235 MHz) δ (ppm) −158.30 (s, 1F). Scan ES⁺ 312 (M+H)⁺. ScanES⁻ 356 (M+HCO₂)⁻.

7l:4-Amino-7-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]pyrrolo[2,3-d]pyrimidine

7l was synthesized from 6g as described for 6a. White lyophilisedpowder. Molecular Formula C₁₃H₁₃FN₄O₃. ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm)3.61 (d, J=5.52 Hz, 1H), 3.63-3.67 (m, 1H), 3.80-3.83 (d, J=12.14 Hz,1H), 3.86-3.88 (d, J=9.38 Hz, 1H), 4.46-4.54 (dd, J=23.23 Hz and J=9.39Hz, 1H), 5.30 (brs, 1H), 6.1 (brs, 1H), 6.41-6.47 (d, J=16.47 Hz, 1H),6.57-6.61 (d, J=16.47 Hz, 1H), 7.04 (s, 2H), 7.37-7.38 (d, J=3.65 Hz,1H), 8.05 (s, 1H) ¹⁹F NMR (DMSO-d₆, 235 MHz) δ (ppm) −157.15 (s, 1F)Scan ES⁺ 293 (M+H)⁺, UV λ_(max) 275 nm

23:9-[(2R)-2-Deoxy-3,5-di-O-isobutyryl-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]guanine

A solution of9-[(2R)-2-Deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-furanosyl]guanine (0.16mmol), 4-dimethylaminopyridine (0.01 mmol), triethylamine (0.48 mmol)and isobutyric anhydride (0.48 mmol), in acetonitrile (1 ml) was stirredat room temperature for 6 hours. The reaction mixture was hydrolysedwith a NaHCO₃ saturated solution. Ethyl acetate was added. The organicphase was separated, washed with NaCl saturated solution, dried overNa₂SO₄, filtered and concentrated under reduced pressure. The crudematerial was purified by flash column chromatography (DCM/EtOH) to yieldthe title compound White powder. Molecular Formula C₂₀H₂₄FN₅O₆ ¹H NMR(DMSO-d₆, 400 MHz) δ (ppm) 1.02-1.22 (m, 12H), 2.53-2.59 (m, 1H),2.65-2.70 (m, 1H), 4.04 (d, J=4.77 Hz, 1H), 4.35-4.40 (m, 3H), 5.88-5.94(dd, J=9.39 Hz and J=8.21 Hz, 1H), 6.21-6.25 (d, J=17.28 Hz, 1H), 6.58(s, 2H), 7.09 (s, 1H), 10.82 (s, 1H). Scan ES⁺ 450.0 (M+H)⁺, UV λ_(max)251 nm

24:N²-Isobutyryl-9-[(2R)-2-deoxy-3,5-di-O-isobutyryl-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]guanine

24 was obtained from the purification of 23. White powder. MolecularFormula C₂₄H₃₀FN₅O₇ ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 1.02-1.22 (m,18H), 2.53-2.59 (m, 1H), 2.65-2.70 (m, 1H), 2.74-2.80 (m, 1H), 4.04 (d,J=4.90 Hz, 1H), 4.35-4.40 (m, 3H), 5.73-5.80 (dd, J=10.14 Hz and J=7.80Hz, 1H), 6.29-6.34 (d, J=17.36 Hz, 1H), 8.23 (s, 1H), 11.80 (brs, 1H),12.3 (brs, 1H). Scan ES⁺ 520 (M+H)⁺, UV λ_(max) 257 nm

4f:5-Fluoro-1-[3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethylsilylethynyl-β-D-arabino-furanosyl]uracile

4f was synthesized from 3f as described for 4a. Orange solid. MolecularFormula C₂₆H₄₅FN₂O₇Si₃ ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 0.13 (s, 9H),0.94-1.06 (m, 28H), 3.75 (m, 1H), 3.96-4.09 (m, 3H), 5.99 (d, J=1.53 Hz,1H), 6.53 (s, 1H), 7.58-7.60 (d, J=6.76 Hz, 1H), 11.8 (brs, 1H) Scan ES⁻599 (M−H)⁻, UV λ_(max) 271 nm

5f:5-Fluoro-1-[(2R)-2-deoxy-2-fluoro-3,5-O-(1,3-diyl-1,1,3,3-tetraisopropyldisiloxane)-2-C-trimethyl-silylethynyl-β-D-erythro-pentofuranosyl]uracile

5f was synthesized from 4f as described for 5a. White solid. MolecularFormula C₂₆H₄₄F₂N₂O₆Si₃. ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 0.13 (s, 9H),0.94-1.06 (m, 28 H), 3.92-3.95 (d, J=12.47 Hz, 1H), 3.96-4.09 (m, 1H),4.21 (d, J=12.22 Hz, 1H), 5.20 (brs, 1H), 6.10-6.15 (d, J=16.53 Hz, 1H),7.56 (s, 1H), 12.23 (brs, 1H) ¹⁹F NMR (DMSO-d₆, 235 MHz) δ (ppm) −160.06(s, 1F), −165.94 (s, 1F) Scan ES⁺ 603 (M−H)⁺, UV λ_(max) 272 nm

6f:5-Fluoro-1-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]uracile

6f was synthesized from 5f as described for 6a. White solid. MolecularFormula C₁₁H₁₀F₂N₂O₅. ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 3.61-3.64 (d,J=12.55 Hz, 1H), 3.81-3.85 (m, 2H), 4.11 (d, J=4.91 Hz, 1H), 4.14-4.20(m, 1H), 5.50 (s, 1H), 6.04-6.09 (d, J=16.91 Hz, 1H), 6.20 (d, J=7.64Hz, 1H), 8.29 (d, J=7.09 Hz, 1H), 12.05 (s, 1H) ¹⁹F NMR (DMSO-d₆, 235MHz) δ (ppm) −158.74 (s, 1F), −166.27 (s, 1F) Scan ES⁺ 289.0 (M−H)⁺, UVλ_(max) 270 nm.

11f:1-[(2R)-2-deoxy-2-C-ethynyl-2-fluoro-β-D-erythro-pentofuranosyl]-5-fluorouracil-5′-yl-bis(S-pivaloyl-2-thioethylphosphate)

11f was synthesized from 6f as described 9k. White solid. MolecularFormula C₂₅H₃₅F₂N₂O₁₀PS₂. ¹H NMR (DMSO-d_(6+D2O), 400 MHz) δ (ppm)1.15-1.17 (m, 18H), 3.10 (t, J=6.40 Hz, 4H), 4-4.08 (m, 5H), 4.19 (d,J=5.39 Hz, 1H), 4.24-4.39 (m, 3H), 6.12 (d, J=16.82 Hz, 1H), 6.39 (d,J=6.07 Hz, 1H), 7.86 (brs, 1H), 12.12 (brs, 1H).

28:9-[(2R)-2,3-dideoxy-2-C-ethynyl-2-fluoro-β-D-glycero-pentofuranosyl]guanine5′-triphosphate sodium

28 was synthesized from 16 as described for 27i. White powder. MolecularFormula C₁₂H₁₂FN₅Na₃O₁₂P₃. ¹H NMR (D₂O, 400 MHz) δ(ppm) 2.61-2.72 (m,2H), 2.95-2.96 (m, 1H), 4.16-4.22 (m, 1H), 4.35-4.40 (m, 1H), 4.6-4.7(m, 1H), 6.17 (d, J=16 Hz, 1H), 8.02 (s, 1H). ¹⁹F NMR (D₂O, 235 MHz) δ(ppm)(−138.95)−(−138.74) (m, 1F), ³¹P NMR (D₂O, 162 MHz) δ (ppm) −10.66(d, J=19.44 Hz, 1P), −11.14 (d, J=19.44 Hz, 1P), −22.82 (t, J=19.44 Hz,1P). Scan ES⁺ 599.6 (M−3Na)³⁺, UV λ_(max) 253 nm.

All publications and patent, applications cited in this specificationare herein incorporated by reference as if each individual publicationor patent application were specifically and individually indicated to beincorporated by reference. While the claimed subject matter has beendescribed in terms of various embodiments, the skilled artisan willappreciate that various modifications, substitutions, omissions, andchanges may be made without departing from the spirit thereof.Accordingly, it is intended that the scope of the subject matter limitedsolely by the scope of the following claims, including equivalentsthereof.

What is claimed is:
 1. A compound of the formula:

or a pharmaceutically acceptable salt, or a stereoisomeric or tautomericform thereof, wherein: R^(y) is optionally substituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, cycloalkyl, cycloalkenyl, hydroxyalkyl, amino,heterocyclyl or heteroaryl; R^(a) and R^(b) are selected as follows: i)R^(a) and R^(b) are each independently hydrogen or optionallysubstituted alkyl, carboxyalkyl, hydroxyalkyl, hydroxyarylalkyl,acyloxyalkyl, aminocarbonylalkyl, alkoxycarbonylalkyl, aryl, arylalkyl,cycloalkyl, heteroaryl or heterocyclyl; or ii) R^(a) and R^(b) togetherwith the nitrogen atom on which they are substituted form a 3-7 memberedheterocyclic or heteroaryl ring; and R¹ is selected from the groupconsisting of ribavirin, viramidine, valopicitabine, PSI-6130, MK-0608,resiquimod and celgosivir, lamivudine, entecavir, telbivudine, racivir,emtricitabine, clevudine, amdoxovir, valtorcitabine, tenofovir andadefovir.
 2. The compound of claim 1 having the formula:

or a pharmaceutically acceptable salt, or a stereoisomeric or tautomericform thereof.
 3. The compound of claim 1 having the formula:

or a pharmaceutically acceptable salt, or a stereoisomeric or tautomericform thereof.
 4. The compound of claim 1 having the formula:

or a pharmaceutically acceptable salt, or a stereoisomeric or tautomericform thereof.
 5. The compound of claim 1 having the formula:

wherein R² and R³ are each independently H, or R² and R³ are linked toform a cyclic group by an alkyl, ester or carbamate linkage; or apharmaceutically acceptable salt, or a stereoisomeric or tautomeric formthereof.
 6. The compound of claim 1 having the formula:

wherein R^(d) is hydrogen, methyl or methoxy; each R^(L) isindependently H, carbamyl, straight chained, branched or cyclic alkyl;acyl; CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substitutedaryl, sulfonate ester, a lipid, an amino acid; an amino acid residue; ora carbohydrate; or a pharmaceutically acceptable salt, or astereoisomeric or tautomeric form thereof.
 7. The compound of claim 1having the formula:

or a pharmaceutically acceptable salt, or a stereoisomeric or tautomericform thereof.
 8. The compound of claim 1, wherein R^(y) is substitutedalkyl; and R^(a) and R^(b) are independently hydrogen, alkyl,substituted alkyl, benzyl or substituted benzyl.
 9. The compound ofclaim 8, wherein R^(y) is hydroxyalkyl or aminoalkyl.
 10. The compoundof claim 1, wherein R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c), where eachR^(c) is independently alkyl, substituted alkyl, aryl or substitutedaryl; and R^(a) and R^(b) are independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl.
 11. The compound of claim 1,wherein R^(a) and R^(b) are independently hydrogen, benzyl orsubstituted alkyl.
 12. The compound of claim 1, wherein R^(y) isselected from the group consisting of alkyl and hydroxyalkyl.
 13. Thecompound of claim 1, wherein R^(y) is —C(CH₃)₂CH₂OH.
 14. The compound ofclaim 1, wherein R² and R³ are each hydrogen, R^(a) is hydrogen, R^(b)is benzyl and R^(y) is —C(CH₃)₂CH₂OH.
 15. The compound of claim 1selected from:


16. The compound of claim 1 having the structure:


17. A pharmecutical composition comprising a compound of claim 1 and apharmaceutically acceptable excipient, carrier or diluent.
 18. Thecomposition of claim 17, wherein the compound is selected from

and pharmaceutically acceptable salts and stereoisomers thereof.
 19. Thecomposition of claim 17, wherein the composition is an oral formulation.20. The composition of claim 18, wherein the composition is an oralformulation.
 21. A kit comprising a combination, wherein the combinationcomprises a compound and ribavirin, wherein the compound has formula:

or a pharmaceutically acceptable salt, or a stereoisomeric or tautomericform thereof, wherein: R^(y) is optionally substituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, cycloalkyl, cycloalkenyl, hydroxyalkyl, amino,heterocyclyl or heteroaryl; R^(a) and R^(b) are selected as follows: i)R^(a) and R^(b) are each independently hydrogen or optionallysubstituted alkyl, carboxyalkyl, hydroxyalkyl, hydroxyarylalkyl,acyloxyalkyl, aminocarbonylalkyl, alkoxycarbonylalkyl, aryl, arylalkyl,cycloalkyl, heteroaryl or heterocyclyl; or ii) R^(a) and R^(b) togetherwith the nitrogen atom on which they are substituted form a 3-7 memberedheterocyclic or heteroaryl ring; and R¹ is selected from the groupconsisting of ribavirin, viramidine, valopicitabine, PSI-6130, MK-0608,resiquimod and celgosivir, lamivudine, entecavir, telbivudine, racivir,emtricitabine, clevudine, amdoxovir, valtorcitabine, tenofovir andadefovir.